Determination of Genetic sex in equine species by analysis of y-chromosomal DNA sequences

ABSTRACT

The present invention relates to DNA sequences, probes and primers specific to the Y chromosome of  Equus caballus . The present invention also relates to methods of determining the sex of a horse, a equine fetus, and equine embryo or equine cells. The present invention further relates to a method for the isolation of Y-chromosomal DNA sequences.

FIELD OF THE INVENTION

The present invention relates to polynucleotide sequences associatedwith the equine Y chromosome and to methods of identifying suchpolynucleotide sequences. The present invention also relates to methodsof determining the primary (i.e. genetic) sex of individuals and ofsamples of cells removed from individuals, and is particularly concernedwith equine sex determination.

BACKGROUND OF THE INVENTION

Many sectors of the various horse industries prefer a preponderance ofanimals of one sex. This may be for reasons of reproductive potential,heritability of particular traits, tractability, performance, statureand physique, appearance or other reasons.

The ability to determine the sex of a fetus is advantageous since itallows optimal management and valuation of pregnancies.

Where methods of assisted reproduction are available, by embryo transfer(with or without induced multiple ovulation) or by recovers and returninto the donor or by in vitro fertilisation, the ability to determinethe sex of an embryo is advantageous since it allows the sex ofpotential progeny to be predetermined. If combined with artificialtwinning by means of embryo bisection (1,2) it further allows enhancedpropagation of the desired sex without reduction in the total number ofpotential progeny.

It would be particularly advantageous to predetermine the sex of progenyby means of insemination of a receptive mare with sperm populationscomprising a preponderance of sperm having one or the other sexchromosome constitution, i.e. either the X chromosome (which sperm yieldfemale progeny) or the Y chromosome (which sperm yield male progeny).Such enriched populations of sperm could also be used to great advantagein in vitro fertilisation. In a further very advantageous application,an individual sperm cell of a known sex chromosome constitution can beinjected into the cytoplasm of a mature oocyte in vitro (ICSI:intra-cytoplasmic sperm injection), effecting fertilisation to yield azygote of known sex. The ability to determine the sex chromosomeconstitution of populations of sperm cells and of individual sperm cellsis an essential prerequisite in such applications.

The primary sex of equine species, as in the overwhelming majority ofmammalian species, is determined by the presence or absence of theentire Y chromosome or a functional portion thereof (3-8). The essentialportion is a gene known as SRY that is responsible for initiating testisdifferentiation (9-11). Secretions of the resultant testis have adominant influence on the development of secondary sex characters (12).

The sex or presumptive sex of an individual horse can thus be determinedby analysis for DNA sequences that are associated uniquely with theequine Y chromosome.

Previous reports of DNA sequences associated with the equine Ychromosome (11,13,14) concern presumptive sequences that are amplifiedby polymerase chain reaction (PCR; 15,16) from primer oligonucleotideswhose sequences are derived from genes known to be Y-linked in othermammalian species, viz. ZFY(13,14) and SRY(11,13). There are nopublished DNA sequence data for DNA sequences associated with the equineY chromosome. Both ZFY and SRY occur in single copy in all mammalianspecies examined (with the known exception of Mus species, in which twosimilar Zfv genes have been described; 17) and so, presumably, in thehorse. In the context of determining the genetic sex of viable embryoswhere only a small number of cells is available from a microscopicbiopsy, assay sensitivity is a significant consideration. The advantagesfor embryo sexing of testing for a DNA sequence that is repeated on theY chromosome have been detailed previously (18,19).

A report of a repeated DNA sequence that is found on the Y chromosome ofhorses (20) concerns a short DNA sequence element known as Bkm(5′-G.A.C/T.A-3′; 21-23) that has been reported in many vertebratespecies. It is also abundant elsewhere in the genome, to the extent thatrepresentatives on the Y chromosome comprise a small minority of thetotal. Such a sequence, of itself, has no utility in the diagnosis ofgenetic sex in microscopic biopsies.

SUMMARY OF THE INVENTION

The present inventors have now identified specific DNA sequences thatare repeated in the Y chromosome of the horse. The nucleic acid isolatescorrespond to all or part of a DNA sequence found on the Y chromosome ofEquus caballus. The present invention therefore provides a number ofpolynucleotide isolates capable of specifically hybridizing to samplesof nucleic acid derived from horses which contain Y chromosomal DNAsequences.

A procedure similar in essence to that used in the first part of thepresent invention has been applied previously to animals where it wasused to observe, but not isolate or otherwise define, DNA fragmentsassociated with the heterogametic sex of chicken (24), cattle (25) andsheep (26).

Accordingly, in a first aspect the present invention provides anisolated polynucleotide, the polynucleotide having a sequence as set outin any one of SEQ ID NOS: 1 to 4 or 8 to 11, or a sequence whichhybridizes thereto.

The polynucleotide sequences of the present invention hybridizespecifically to the equine Y chromosome. By “hybridize specifically tothe equine Y chromosome” we mean the polynucleotides hybridize to arepeat sequence which is present on the equine Y chromosome in asubstantially greater copy number than is present elsewhere in theequine genome. Preferably, the sequence is present in less than sixcopies and more preferably in only one copy in the haploid femalegenome.

In a preferred embodiment the polynucleotide sequence has a sequence asset out in SEQ ID NO: 3 or a sequence which hybridizes thereto.

The polynucleotide sequences of the present invention preferablyhybridize to sequences set out in SEQ ID NOS: 1 to 4 or 8 to 11 underhigh stringency. When used herein, “high stringency” refers toconditions that (i) employ low ionic strength and high temperature forwashing after hybridization, for example, 0.1×SSC and 0.1% (w/v) SDS at50° C.; (ii) employ during hybridization conditions such that thehybridization temperature is 25° C. lower than the duplex meltingtemperature of the hybridizing polynucleotides, for example 1.5×SSPE,10% (w/v) polyethylene glycol 6000 (27), 7% (w/v) SDS (28), 0.25 mg/mlfragmented herring sperm DNA at 65° C.; or (iii) for example, 0.5Msodium phosphate, pH 7.2. 5 mM EDTA. 7% (w/v) SDS (28) and 0.5% (w/v)BLOTTO (29.30) at 70° C.: or (iv) employ during hybridization adenaturing agent such as formamide (31), for example, 50% (v/v)formamide with 5×SSC, 50 mM sodium phosphate (pH 6.5) and 5×Denhardt'ssolution (32) at 42° C.; or (v) employ, for example, 50% (v/v)formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% (w/v) sodiumpyrophosphate, 5×Denhardt's solution (32). Sonicated salmon sperm DNA(50 μg/ml) and 10% dextran sulphate (33) at 42° C. See generallyreferences 34-36.

In a further preferred embodiment, the polynucleotide which hybridisesunder stringent conditions is less than 500 nucleotides, more preferablyless than 200 nucleotides, and more preferably less than 100 nucleotidesin length.

In a further preferred embodiment, the polynucleotide sequences of thepresent invention share at least 40% homology, more preferably at least60% homology, more preferably at least 80% homology, more preferably atleast 90% homology and more preferably at least 95% homology with asequence shown in any one of SEQ ID NOS: 1 to 4 or 8 to 11, wherein thehomology is calculated by the BLAST program blastn as described inAltschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,Miller, W. And Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs”, Nucleic Acids Research25(17):3389-3402.

In a further preferred embodiment, the polynucloetide sequence of thepresent invention hybridises under stringent conditions to a sequencecharacterised by nucleotides 990-2497 of SEQ ID NO: 8, 421-1920 of SEQID NO. 9, 421-1930 of SEQ ID NO. 10, or 1502-2996 of SEQ ID NO. 11.

The polynucleotide of the present invention may comprise DNA or RNAsequences.

The present invention also provides a vector including a polynucleotidesequence according to the first aspect of the present invention and ahost cell transformed with such a vector.

In a second aspect, the present invention provides an oligonucleotideprobe or primer of at least 8 nucleotides, the oligonucleotide having asequence that hybridizes to a polynucleotide of the first aspect of thepresent invention.

In a preferred embodiment the oligonucleotide is at least 10, morepreferably at least 15 and more preferably at least 18 nucleotides inlength.

In one preferred embodiment the oligonucleotide is derived from thesequence shown in SEQ ID NO:3. In one preferred embodiment theoligonucleotide comprises the sequence:

5′-AGCGGAGAAAGGAATCTCTGG-3′ (SEQ ID NO: 12) or

5′-TACCTAGCGCTTCGTCCTCTAT-3′ (SEQ ID NO: 13) derived from nts 6-26 andthe reverse complement of nts 184-205, respectively, of the equine malegenomic DNA sequence shown in SEQ ID NO: 7.

It will be appreciated that the probes or primers of the presentinvention may be produced by in vitro or in vivo synthesis. Methods ofin vitro probe synthesis include organic chemical synthesis processes orenzymatically mediated synthesis, e.g. by means of SP6 RNA polymeraseand a plasmid containing a polynucleotide sequence according to thefirst aspect of the present invention under transcriptional control ofan SP6 specific promoter.

In a further preferred embodiment the oligonucleotide probe isconjugated with a label such as a radioisotope, an enzyme, biotin, afluorescer or a chemiluminescer.

In a third aspect, the present invention provides a method ofdetermining the sex of a horse, an equine fetus, an equine embryo or anequine cell(s) which method includes analysing a biological samplederived from the horse or the fetus or embryo or the population ofcells, for the presence of a polynucleotide according to the firstaspect of the present invention.

The equine cell(s) may be, for example, the sperm cells of a horse. In apreferred embodiment they may be populations of sperm cells orindividual sperm cells that have been resolved by flow cytometry afterstaining with the fluorescent DNA-binding dye Hoechst 33342 (37,38).

The equine cell(s) may further be, for example, nucleated fetal cells.Such cells may be collected by amniocentesis or chorionic villussampling. In a preferred embodiment they may be sampled in theperipheral blood of a pregnant mare (see generally reference 39 thedisclosure of which is incorporated herein by reference).

In order to minimise the possibility of false negatives, the method ispreferably conducted with one or more suitable positive controls. Forexample, the biological sample may be simultaneously analysed for thepresence of a sequence which is present in approximately equal copynumbers in male and female horses. The biological sample may beanalysed, for example, for the presence of a dispersed autosomalrepeated sequence.

It will be understood by a person skilled in this field that an analysisto determine whether a sample contains the polynucleotide sequence ofthe present invention may be performed in a number of ways. For example,the analysis may involve Southern blot hybridization, dot blothybridization or in situ hybridization tests using probes according tothe present invention. Alternatively, the analysis may involve thetechnique of polymerase chain reaction (PCR; 16) or ligationamplification reaction (LAR: 40,41) using oligonucleotide primers andprobes of the present invention.

The term “polymerase chain reaction” or “PCR” when used herein generallyrefers to a procedure where minute amounts of a specific piece ofnucleic acid, RNA and/or DNA, are amplified as described in references42 and 43. Generally, sequence information from the ends of the regionof interest or beyond needs to be available, such that oligonucleotideprimers can be designed; these primers will be identical in sequence orsimilar in sequence to opposite strands of the template to be amplified.The 5′ terminal nucleotides of the two primers may coincide with theends of the amplified material. PCR can be used to amplify specific RNAsequences, specific DNA sequences from total genomic DNA, and cDNAtranscribed from total cellular RNA, bacteriophage or plasmid sequences,etc. See generally references 16 and 44.

As used herein, PCR is considered to be one, but not the only, exampleof a nucleic acid polymerase reaction method for amplifying a nucleicacid test sample, comprising the use of an established nucleic acid (DNAor RNA) as a primer, and utilises a nucleic acid polymerase to amplifyor generate a specific piece of nucleic acid or to amplify or generate aspecific piece of nucleic acid which is complementary to a particularnucleic acid (see, for example, references 45 and 46).

The terms “ligation chain reaction” or “LCR” or “ligation amplificationreaction” or “LAR” when used herein generally refer to a procedure whereminute amounts of a specific piece of nucleic acid, RNA and/or DNA, areamplified as described in references 40 and 41. Generally, sequenceinformation from the region of interest needs to be available, such thatoligonucleotide pairs can be designed that are complementary to adjacentsites on an appropriate nucleic acid template. The oligonucleotide pairis ligated together by the action of a ligase enzyme. The amount ofligated product may be increased by either linear or exponentialamplification using sequential rounds of such template-dependentligation. In the case of linear amplification, a single pair ofoligonucleotides is ligated, the reaction is heated to dissociate theligation product from its template, and similar additional rounds ofligation are performed. Exponential amplification utilises two pairs ofoligonucleotides, one pair being complementary to one strand of a targetsequence and the other pair being complementary to the second strand ofthe target sequence. In this case the products of ligation serve asmutually complementary templates for subsequent rounds of ligation,interspersed with heating to separate the ligated products from thetemplate strands. A single base-pair mismatch between the annealedoligonucleotides and the template prevents ligation, thus allowing thedistinction of single base-pair differences between DNA templates. LARcan be used to amplify specific RNA sequences, specific DNA sequencesfrom total genomic DNA, and cDNA transcribed from total cellular RNA,bacteriophage or plasmid sequences, etc. See generally references 40 and41. As used herein, LAR is considered to be one, but not the only,example of a nucleic acid ligase reaction method for amplifying anucleic acid test sample, comprising the use of an established nucleicacid (DNA or RNA) as a primer/probe, and utilises a nucleic acid ligaseto amplify or generate a specific piece of nucleic acid or to amplify orgenerate a specific piece of nucleic acid which is complementary to aparticular nucleic acid (see, for example, references 47 and 48).

In a fourth aspect, the present invention provides a kit for sexdetermination of a horse, an equine fetus, an equine embryo, an equinecell or a population of equine cells, which kit includes apolynucleotide according to the first aspect of the present invention oran oligonucleotide probe or primer according to the second aspect of thepresent invention.

The terms “EY.AC6”, “EY.AD11”, “EY.AI5” and “EY.AM7” as used hereinrefer to, where provided, the specific DNA sequences set forth in SEQ IDNOS: 1-4 respectively. These terms also include variants wherenucleotides have been substituted, added to or deleted from the relevantsequences shown in SEQ ID NOS: 1-4 so long as the variants hybridizespecifically to the equine Y chromosome.

Such variants may be naturally occurring variants which may arise withinan individual or a population by virtue of point mutation(s),deletion(s) or insertion(s) of DNA sequences, by recombination, geneconversion, flawed replication or rearrangement. Alternatively, suchvariants may be produced artificially, for example by site-directedmutagenesis, by “gene shuffling”, by deletion using exonuclease(s)and/or endonuclease(s), or by the addition of DNA sequences by ligatingportions of DNA together, or by the addition of DNA sequences bytemplate-dependent and/or template-independent DNA polymerase(s).

The EY.AC6 DNA sequence is shown in SEQ ID NO: 1. The sequence,comprising 432 base pairs of nucleotides, was determined from a fragmentof DNA that was cloned into plasmid pGEM-T (trademark Promega). Thecloned fragment had been recovered from a polyacrylamide gel followingelectrophoresis and staining of the products of RAPD PCR of male equinegenomic DNA with Operon (trademark) primer OPAC.06. The fragment wasselected because it was visible as a product of RAPD PCR of male but notfemale genomic DNA. Homologues of the cloned fragment EY.AC6 have beenshown, by its hybridization to Southern blots of genomic DNA from maleand female Equus caballus, to be present in both sexes but are repeatedat much higher amounts in males, with the haploid female genomecontaining just one or a small number of copies. The defined sequenceEY.AC6 appears to be contiguous with sequence EY.AM7 in the equine Ychromosome since the two sequenced isolates share a region of overlap of128 bp with 91% similarity.

The EY.AD11 DNA sequence is shown in SEQ ID NO: 2. The sequence,comprising 600 base pairs of nucleotides, was determined from a fragmentof DNA that was cloned into plasmid pGEM-T (trademark Promega). Thecloned fragment had been recovered from a polyacriylamide gel followingelectrophoresis and staining of the products of RAPD PCR of male equinegenomic DNA with Operon (trademark) primer OPAD.11. The fragment wasselected because it was visible as a product of RAPD PCR of male but notfemale genomic DNA. Homologues of the cloned fragment EY.AD11 have beenshown, by its hybridization to Southern blots of genonlic DNA from maleand female Equus caballus, to be present in both sexes but are repeatedat much higher amounts in males, with the haploid female genomecontaining just one or a small number of copies.

The EY.AI5 DNA sequence is shown in SEQ ID NO: 3. The sequence,comprising 230 base pairs of nucleotides, was determined from a fragmentof DNA that was cloned into plasmid pGEM-3Z (trademark Promega). Thecloned fragment had been recovered from a polyacrylamide gel followingelectrophoresis and staining of the products of RAPD PCR of male equinegenomic DNA with Operon (trademark) primer OPAI.05. The fragment wasselected because it was visible as a product of RAPD PCR of male but notfemale genomic DNA. Homologues of the cloned fragment EY.AI5 have beenshown, by its hybridization to Southern blots of genomic DNA from maleand female Equus caballus, to be present in both sexes but are repeatedat much higher amounts in males, with the haploid female genomecontaining just one or a small number of copies.

The EY.AM7 DNA sequence is shown in SEQ ID NO: 4. The sequence,comprising 285 base pairs of nucleotides, was determined from a fragmentof DNA that was cloned into plasmid pGEM-T (trademark Promega). Thecloned fragment had been recovered from a polyacrylamide gel followingelectrophoresis and staining of the products of RAPD PCR of male equinegenomic DNA with Operon (trademark) primer OPAM.07. The fragment wasselected because it was visible as a product of RAPD PCR of male but notfemale genomic DNA. Homologues of the cloned fragment EY.AM7 have beenshown, by its hybridization to Southern blots of genomic DNA from maleand female Equus caballus, to be present in both sexes but are repeatedat much higher amounts in males, with the haploid female genomecontaining just one or a small number of copies. The defined sequenceEY.AM7 appears to be contiguous with sequence EY.AC6 in the equine Ychromosome since the sequences isolated share a region of overlap of 128bp with 91% similarity.

The DNA sequences described herein (SEQ ID NOS: 1-4) were determined bychain-termination DNA sequencing techniques (49) usingfluorescence-labelled dideoxynucleotides (50-53).

It will be appreciated by those skilled in the art that thepolynucleotide sequences of the present invention are advantageous inthat they are present in multiple copies on the Y chromosome, therebyproviding greater sensitivity in assays for the presence of a Ychromosome than is possible when the assay involves detection of aunique (single copy) DNA sequence. This allows detection to be appliedwith greater facility to very small samples, as in a few cells removedfrom a viable embryo (2) or cells of fetal origin in peripheral blood ofa pregnant mare (39) or sperm cells separated by fluorescence activatedcell sorting (38).

The polynucleotide sequences and oligonucleotide primers and probes ofthe present invention have application, for example, in sexing of embryobiopsy; fetal sex detection, i.e. by amniocentesis, chorionic villussampling, fetal cells circulating in peripheral blood of a pregnantmare; analysis of the sex chromosome constitution of an individual spermcell or of populations of sperm cells; resolution of ambiguities insexual phenotype; sex analysis of tissues derived from horses (meat,hide, hair, bone, etc. from living or dead horses); and similarapplications in related equine species, including extinct or endangeredspecies.

The polynucleotide sequences and oligonucleotide primers and probes ofthe present invention also have a variety of uses in addition to theiruse in sexual identification. For example, the sequences may be used toscreen recombinant DNA libraries prepared from a variety of mammalianspecies. The DNA sequences may be used to deduce similar sequences orgenetically linked sequences having similar functionality. The sequencesmay also be used in chromosome walking or jumping techniques to isolatecoding and non-coding sequences proximal to the nucleotide sequence ofthe present invention.

According to a further aspect of the present invention, there isprovided a method for the isolation of Y-chromosomal DNA sequencescomprising:

pooling equivalent amounts of genomic DNA from a number of male animalsof a single species and pooling equivalent amounts of genomic DNA from asimilar number of female animals of the same species, with the femaleanimals preferably being related closely to the male animals, e.g.siblings;

subjecting equivalent samples of the male and female pooled DNA mixturesto PCR with an arbitrary oligonucleotide primer and resolving theresultant fragments by gel electrophoresis;

examining the stained resolved products for fragments that are amplifiedfrom male DNA but not from female DNA;

recovering said fragment(s) from an electrophoresis gel and isolatingindividual fragments by molecular cloning; and

PCR analysis of samples of male and female genomic DNA usingoligonucleotide primers derived from the DNA sequence of said isolatedfragment(s).

In a preferred embodiment the method includes the additional step afterstep (iii) of confirming the male association of fragment(s) by PCR andelectrophoretic analysis of equivalent genomic DNA samples from a numberof individual male and female animals. Preferably the method alsoincludes an additional step after step (iv) of confirming the isolationof individual male-associated fragment(s) by hybridization of thelabelled said fragment(s) with samples of male and female genomic DNA.

The terms “comprise”, “comprises” and “comprising” as used throughoutthe specification are intended to refer to the inclusion of a statedcomponent or feature or group of components or features with or withoutthe inclusion of a further component or feature or group of componentsor features.

The present invention will now be described, by way of example only,with reference to the following non-limiting drawings and examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows hybridization analysis of horse breeds, donkey and camelwith male-associated sequence EY.AI5. Samples of genomic DNA (2.5 μg)from male (m) and female (f) horses of various breeds (Family Equidae:Equus caballus) as well as Przewalski's horse (E. przewalskii), donkey(E. asinus) and the camel (Family Camelidae: Camelus dromedarius) asindicated, were digested with Sau3AI. The fragments were resolved byagarose gel electrophoresis, transferred onto positively-charged nylonmembrane and hybridized with digoxigenin-labelled probe EY.AI5, asdescribed in the text. The lane labelled M contained DNA standards whosesizes are indicated in base pairs.

FIG. 2 shows the sequence of fragments amplified directly from genomicDNA of male and female horses using primers EQYL1 and EQYR1. Differencesbetween the sequences determined from male and female genomic DNA areindicated by *; differences between the cloned EY.AI5 sequence (SEQ IDNO: 3) and the sequence from male genomic DNA are indicated by *. Theunderlined region from nt 6 to nt 26 is the sequence of sexing primerEQYL2; the underlined region from nt 184 to nt 205 is the reversecomplement of the sequence of sexing printer EQYR5 (refer to text).

FIG. 3 shows hybridization analysis of recombinant phage DNA with clonedmale-associated sequences. Samples of DNA (10 μg) of Lambda Fix® IIvectors containing equine genomic inserts were digested with restrictionenzymes EcoRI and HindIII as shown. Digests were treated at 68° C. for15 min then resolved by agarose gel electrophoresis before transfer topositively-charged nylon membrane as described in the text. The membranewas hybridized with digoxigenin-labelled probes prepared by PCRamplification of cloned inserts from the flanking plasmid primers SP6and T7. One probe was stripped from the membrane by methods described inthe text before hybridization with the second probe. The inserts used asprobes were: (a) EY.AI5; (b) EY.AD11. A photograph of the gel takenunder uv transillumination before DNA transfer is shown. The laneslabelled M contained DNA standards whose sizes are indicated in basepairs.

FIG. 4 shows in (a) the sites for restriction enzyme EcoRI in the equinegenomic DNA insert 32.3 after excision of the insert, together with itsflanking T3 and T7 promoter sequences, from the Lambda Fix® II vectorwith the restriction enzyme NotI. The position of 4.7 kb subcloned EcoRIfragment 32.3E5 is indicated. The complete sequence of 32.3E5 wasdetermined and, in (b), the positions of previously described sequencesEY.AC6, EY.AD11, EY.AI5 and EY.AM7 within the subclone are illustrated,as is the relative position of the truncated LINE repeat EY.LINE asdefined in the text. There is a close relationship between the DNAsequences of subclone 32.3E5 and subclone 33.1H7 (see FIG. 5) whichallows 33.1H7 to be superimposed on 32.3E5 as shown.

FIG. 5 shows in (a) the sites of restriction enzyme HindIII in thegenomic DNA insert 33.1 after excision of the insert, together with itsflanking T3 and T7 promoter sequences, from the Lambda Fix® II vectorwith the restriction enzyme NotI. The locations of two 3.4 kb subclonedrepeated HindIII fragments 33.1H7 and 33.1H2 are indicated although itwas not possible to determine which repeat occupied either of the twopossible positions. The complete sequences of both fragments weredetermined and found to have 88-90% identity. In (b). the positions ofpreviously described sequences EY.AC6, EY.AD11, EY.AI5 and EY.AM7 withinthe subclone 33.1H7 are illustrated, as is the relative position of thetruncated LINE repeat EY.LINE as defined in the text. There is a closerelationship between the DNA sequences of subclone 32.3E5 and subclone33.1H7 which allows 33.1H7 (and 33.1H2) to be superimposed on 32.3E5 asshown in FIG. 4b.

FIG. 6 shows in (a) the sites of restriction enzymes EcoRI and HindIIIin the equine genomic DNA insert 36.1 after excision of the insert,together with its flanking T3 and T7 promoter sequences, from the LambdaFix® II vector with the restriction enzyme NotI. The position of 6.0 kbsubcloned EcoRI fragment 36.1E2 is indicated, as is the position of 4.4kb subcloned HindIII fragment 36.1H7. The complete sequence of 36.1H7was determined and, in (b), the positions of previously describedsequences EY.AC6, EY.AD11, EY.AI5 and EY.AM7 within the subclone areillustrated, as is the relative position of the truncated LINE repeatEY.LINE as defined in the text. There is a close relationship betweenDNA sequences from base 1378 to base 4355 of subclone 36.1H7 tosequences in subclone 32.3E5 (see FIG. 4b) and subclones 33.1H7 and33.1H2 (see FIG. 5b). Sequence in subclone 36.1H7 located 5′ to thishomologous region encoded inverted and direct repeats of EY.AC6, EY.AD11and intervening sequence.

BRIEF DESCRIPTION OF SEQUENCE LISTINGS

SEQ ID NO: 1 shows the sequence of one strand of equine repeat elementEY.AC6 comprising 432 complementary base pairs. The sequence is writtenin single-letter code from the 5′-terminus to the 3′-terminus accordingto standard practice.

SEQ ID NO: 2 shows the sequence of one strand of equine repeat elementEY.AD11 comprising 600 complementary base pairs.

SEQ ID NO: 3 shows the sequence of one strand of equine repeat elementEY.AI5 comprising 230 complementary base pairs.

SEQ ID NO: 4 shows the sequence of one strand of equine repeat elementEY.AM7 comprising 285 complementary base pairs.

SEQ ID NO: 5 shows the sequence of the cloned EY.AI5 sequence

SEQ ID NO: 6 shows the sequence of fragments amplified directly fromgenomic DNA of female horses using primers EQYL1 and EQYR1.

SEQ ID NO: 7 shows the sequence of fragments amplified directly fromgenomic DNA of male horses using primers EQYL1 and EQYR1.

SEQ ID NO: 8 shows the sequence of one strand of subclone 32.3E5comprising 4693 complementary base pairs of equine genomic DNA. Subclone32.3E5 is an EcoRI fragment of recombinant phage 32.3. The position ofthe fragment within the phage insert is shown in FIG. 4a.

SEQ ID NO: 9 shows the sequence of one strand of subclone 33.1H7comprising 3430 complementary base pairs of equine genomic DNA. Subclone33.1H7 is one of two repeated HindIII fragments of recombinant phage33.1 and is 88-90% homologous with a second HindIII fragment, 33.1H2(detailed in SEQ ID NO:10). The position of the repeated fragment withinthe phage insert is shown in FIG. 5a.

SEQ ID NO: 10 shows the incomplete sequence of one strand of subclone33.1H2 comprising 3230 complementary base pairs of equine genomic DNA,from 1 to 2122 and from 2342 to 3450 of the subclone. Subclone 33.1H2 isthe second of two repeated HindIII fragments of phage 33.1 and is 88-90%homologous with the HindIII fragment, 33.1H7 (detailed in SEQ ID NO: 9).The position of the repeated fragment within the phage insert is shownin FIG. 5a.

SEQ ID NO: 11 shows the sequence of one strand of subclone 36.1H7comprising 4355 complementary base pairs of equine genomic DNA. Subclone36.1H7 is a HindIII fragment of phage 36.1. The position of the fragmentwithin the phage insert is shown in FIG. 6a.

SEQ ID NO: 12 shows an oligonucleotide probe (EQYL2) derived from SEQ IDNO:3.

SEQ ID NO: 13 shows an oligonucleotide probe (EQYR5) derived from SEQ IDNO:3.

SEQ ID NO: 14 shows an oligonucleotide primer (EQYR4) derived from SEQID NO:3.

SEQ ID NO: 15 shows an oligonucleotide primer (EQYL1) derived from SEQID NO:3.

SEQ ID NO: 16 shows an oligonucleotide primer (EQYR1) derived from SEQID NO:3.

SEQ ID NO: 17 shows an oligonucleotide primer (EQSIN8) derived from SEQID NO:3.

SEQ ID NO: 18 shows all oligonucleotide primer (EQSIN9) derived from SEQID NO:3.

SEQ ID NO: 19 shows an oligonucleotide primer (mEQYL2) derived from SEQID NO:3.

SEQ ID NO: 20 shows an oligonucleotide primer (mEQYR5) derived from SEQID NO: 3.

SEQ ID NO: 21 shows an oligonucleotide primer (mEQSIN8) derived from SEQID NO:3.

SEQ ID NO: 22 shows an oligonucleotide primer (mEQSIN9) derived from SEQID NO:3.

Definitions and Abbreviations

ATP adenosine-5′-triphosphate

BLOTTO skim milk powder

bp base pairs

ccc covalently closed circular

cfu colony-forming units

BSA bovine serum albumin

Denhardt's solution 0.02% (w/v) BSA, 0.02% (w/v) Ficoll 400, 0.02%

(w/v) PVP

DIG digoxigenin

DNA deoxyribonucleic acid

dNTP deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP)

DTT dithiothreitol

EDTA ethylenediaminetetraacetic acid

g force of gravity

h hour(s)

LAR ligation amplification reaction

LB Luria-Bertani

mg milligram(s)=10⁻³ gram

min minute(s)

ml milliliter(s)=10⁻³ liter

μg microgram(s)=10⁻⁶ gram

μl microliter(s)=10⁻⁶ liter

ng nanogram(s)=10⁻⁹ gram

nm nanometer=10⁻⁹ meter (ref. wavelength of light)

nt(s) nucleotide(s)

oligonucleotide single-stranded DNA<30 nts

PAGE polyacrylamide gel electrophoresis

PBS phosphate-buffered saline=100 mM NaCl, 2.7 mM KCl, 1.75 mM

KH₂PO₄, 4.3 mM Na₂HPO₄, pH 7.4

PCR polymerase chain reaction

pg picogram(s)=10⁻¹² gram

polynucleotide single- or double-stranded DNA or RNA

primer oligonucleotide used to prime PCR

probe (labelled) nucleic acid that hybridizes to specific targetsequence(s)

PVP polyvinylpyrrolidone

RAPD random amplification of polymorphic DNA

RNA ribonucleic acid

rpm revolutions per minute

SDS sodium dodecylsulphate

SINE short interspersed repetitive element

SSC standard saline-citrate=0.15 M NaCl, 15 mM trisodium citrate

SSPE standard saline-phosphate-EDTA=0.18M NaCl, 10 mM NaH₂PO₄,

1 mM EDTA, pH 7.7

TAE tris-acetate-EDTA=40 mM tris-acetate, 2 mM acetic acid, 10 mM EDTA,pH 8.4

Taq Thermus aquaticus

TBE tris-borate-EDTA=89 mM tris-HCl, 0.89M sodium borate, 2 mM EDTA, pH8.4

TE tris-EDTA=10 mM tris-HCl, 1 mM EDTA, pH 7.5

TEMED N,N,N′,N′-tetramethylethylenediamine

temp temperature

tris tris(hydroxymethyl)-aminomethane

uV ultraviolet

V volts

vol volume equivalent

v/v volume/volume equivalent

DETAILED DESCRIPTION OF THE INVENTION Example 1

Preparation Of Genomic DNA From Equine Blood Samples: Equine bloodsamples were collected into 10 ml EDTA Vacutainers®, placed immediatelyon ice and delivered to the laboratory within two days. It was foundthat samples could be stored in a Vacutainer® at 4° C. for up to sixmonths without significant loss of yield or quality of DNA extractedtherefrom.

Twenty five ml of cold lysis buffer (0.32M sucrose, 10 mM tris-HCl, pH7.5, 5 mM MgCl₂, 1% (v/v) Triton X-100) was added to 10 ml of wholeblood. The suspension was centrifuged at 4000×g for 20 min at 4° C. andthe pelleted cells were resuspended in PBS and recentrifuged. The cellswere then suspended in 9 ml of TE. The suspension was adjusted to 25 mMEDTA, 0.5% (w/v) SDS and 0.1 mg/ml of proteinase K (Boehringer Mannheim)and the lysed mixture was incubated overnight at 37° C. with gentleagitation. The digested sample was extracted with 5 ml ofphenol/chloroform (equal volumes of phenol equilibrated withtris-HCl/EDTA (Sigma) and 24:1 (v/v) chloroform/isoamyl alcohol) for 60min and the mixture was centrifuged at 4000×g for 25 min at 25° C. Theaqueous phase was removed from each tube and transferred to a cleantube.

DNA was precipitated by the addition of 2.5 vol ethanol or 1 volpropanol, the supernatant decanted and the DNA pellet rinsed with 0.5 mlof 70% (v/v) ethanol and air-dried. The DNA was finally dissolved in 2ml of 0.1×TE and stored at −20° C.

DNA concentrations were determined using a Pharmacia Gene Quant RNA/DNAcalculator. The yield was typically 50-250 μg of high molecular weightDNA (estimated by ethidium staining after agarose gel electrophoresis).

Conceptual Basis for Identification of Male-Associated DNA

The Y chromosome is the sole genetic difference between male and femalehorses, being present in all nucleated cells of normal males and absentfrom the cells of normal females. This genetic difference must bereflected in the presence of Y-chromosomal DNA sequences in the malegenome that are absent from the female genome. It would be expected thatY-chromosomal, male-specific DNA sequences could be identified by atechnique that surveys multiple genomic DNA sequences at random (54), bycomparing survey data from normal male and female genomes which are inall other respects identical.

It was not possible to obtain isogenic male and female horses, i.e.individuals whose genomes are identical except for the Y chromosome ofthe male (cf. inbred strains of mice). In the absence of genetichomology, a combination of statistical and genetic techniques was usedto generate pseudo-isogenic samples of male and female equine DNA.

DNA was extracted from white blood cells of nine brother-sister siblingpairs and equal amounts of DNA from each of the nine males were pooledto provide a sample of male DNA. Equal amounts of DNA from each of theirsisters were pooled to provide a parallel sample of pseudo-isogenicfemale DNA.

RAPD PCR Of Pooled DNA Samples

The pooled mixtures of male and female DNA were surveyed formale-associated sequence differences by PCR amplification, usingdecanucleotide primers known as RAPD primers that are availablecommercially from Operon Technologies.

The method used for RAPD PCR was adapted from a method describedpreviously (55). Each PCR reaction contained 25 ng of equine genomicDNA, 5 μM RAPD primer (Operon Technologies), 3 units of Taq DNApolymerase Stoffel fragment (Perkin-Elmer), 200 μM of each of the fourdNTPs (Promega), 10 mM tris-HCl, pH 8.0, 10 mM KCl and 5 mM MgCl₂ in atotal volume of 20 μl.

Reactions were cycled in a Corbett Research PC-960 Air-CooledThermocycler with an initial step of 94° C. for 5 min followed by 35cycles consisting of 94° C. for 30 sec then 1 min at each of 57° C., 56°C., 55° C., 54° C. and 53° C.; on completion of cycling the samples wereheated at 72° C. for 5 min.

Electrophoretic Analysis of RAPD PCR Products

Polyacrylamide gel electrophoresis was used to resolve the products ofRAPD PCR, greatly increasing the resolution of fragments relative tothat attainable by agarose gel electrophoresis. Silver staining enhancedthe sensitivity of detection compared with uv fluorescence of ethidiumbromide.

DNA amplification products were resolved by polyacrylamide gelelectrophoresis (PAGE) in a Bio-Rad Mini-Protean II. The polyacrylamidegels were 10% (w/v) acrylamide and 2% (w/v) bis-acrylamide in TBE buffercontaining 10% (w/v) urea and 5% (v/v) glycerol. Ammonium persulphate(0.15% w/v) and TEMED (0.15% v/v) were used to initiate and catalysepolymerisation.

The 0.5 mm gels were cast on Gel Bond PAG backing film (FMC; 56).Samples (2 μl) of PCR reaction product were mixed with 1 μl of loadingbuffer (40% (w/v) urea, 3% (w/v) Ficoll 400, 10 mM tris-HCl, pH 8.0, 3mM EDTA, 0.02% (w/v) xylene cyanol, 0.02% (w/v) bromophenol blue),loaded into pre-formed slots and electrophoresed in TBE buffer at 300Vfor 40 min. Resolved DNA fragments were visualised by silver staining(57).

In total, 216 different Operon RAPD primers were used to screen thepooled pseudo-isogenous samples of male and female DNA. of which 90%yielded clear, reproducible results for both pooled samples.

Identification of Male-Associated DNA Fragments

Nineteen of the 216 tested primers were found to amplify a fragment fromthe male DNA pool that was either less intense than a fragment ofsimilar size in the female DNA pool or apparently absent from the PCRproducts of the female DNA pool. To determine whether candidatefragments were indeed amplified from the DNA of all males and onlymales, primers yielding candidate male-associated fragments from thepooled DNA samples were used for RAPD PCR of DNA isolated from a numberof individual males and females. A fragment amplified differentiallyfrom pooled male DNA could arise from an autosomal polymorphism in oneor two individuals, a possibility confirmed by the occasionalobservation of differential RAPD PCR fragments from the pooled femaleDNA sample.

The 19 candidate primers were used to amplify individual DNA samplesfrom four male and four female horses. Unambiguous male-associatedfragments were evident in the products from five of these primers:OPAC.06 (5′-CCAGAACGGA-3′ (SEQ ID NO:23)), OPAD.11 (5′-CAATCGGGTC-3′(SEQ ID NO:24)), OPAI.05 (5′-GTCGTAGCGG-3′ (SEQ ID NO:25)), OPAM.01(5′-TCACGTACGG-3′ (SEQ ID NO:26)) and OPAM.07 (5′-AACCGCGGCA-3′ (SEQ IDNO:27)). The sizes of the differential fragments were estimated atapproximately 460 bp, 530 bp, 240 bp, 320 bp and 300 bp, respectively.

Isolation of Male-Associated DNA Fragments

For each of the five candidate male-specific fragments, a slicecontaining the fragment was cut from the silver-stained polyacrylamidegel and allowed to stand in 20-50 μl of 0.1×TE at room temp for 60 min.Eluted DNA was re-amplified under the conditions described above forRAPD PCR using the relevant RAPD primer and 1 μl of excised fragmentsolution as template. Reactions were cycled in a Corbett Research PC-960Air-Cooled Thermocycler with an initial step at 94° C. for 2 minfollowed by 35 cycles of 94° C. for 30 sec then 55° C. or 60° C. for 1min; on completion of cycling the samples were heated at 72° C. for 2min.

It was necessary to confirm that the re-amplified DNA samplescorresponded in electrophoretic mobility with the candidate RAPDfragments and that each contained a male-associated DNA fragment.Accordingly, each re-amplified sample was electrophoresed on apolyacrylamide gel adjacent to the products of PRAP PCR of male andfemale genomic DNA with the relevant primer, and with the products ofRAPD PCR from female DNA mixed with the re-amplified sample.

In each case, the re-amplified fragment migrated similarly to thefragment associated differentially with male DNA. In furtherconfirmation, each of the re-amplified samples was labelled withdigoxigenin and the resultant probes were hybridized to Southern blotsof male and female horse genomic DNA that had been digested with therestriction enzyme Sau3AI. Each re-amplified fragment showed anunequivocal male-associated pattern of hybridization (data not shown;refer to “Colony Screening By Dot Blot Hybridization” for details ofprobe preparation, hybridization and detection). In each case the probealso hybridized with female genomic DNA, implying that the fragments maynot be associated uniquely with the Y chromosome and/or the sampleincluded contaminating non-Y-chromosomal DNA.

Re-amplified PCR products were electrophoresed in 1% (w/v) LMP agarose(Sigma) in 0.5×TBE buffer. The material recovered from PCR with theOPAI.05 primer was visualised by illumination at 302 nm of an ethidiumbromide-stained gel. The material recovered from PCR of equine genomicDNA with the OPAC.06, OPAD.11, OPAM.01 and OPAM.07 primers werevisualised by staining with crystal violet (58).

A minimal portion of gel containing the desired fragment was excised andmelted at 70° C. in a 1.5 ml microcentrifuge tube. The molten gel slicewas diluted with three volumes of TE and extracted with an equal volumeof phenol (saturated with TE) at 70° C. for 2 mill. The tube wastransferred to ice for 2 min then centrifuged at 14,000 rpm in anEppendorf 5414C microcentrifuge for 4 min at room temp and the aqueousphase removed into a clean tube. The phenol phase was back-extractedwith 50 μl of TE and this was combined with the original extractedaqueous phase.

DNA was precipitated by the addition of 0.1 vol of 3M sodium acetate. pH5. and 2.5 vol ethanol. The tube was stored overnight at −20° C. thencentrifuged at 13000 rpm in an Eppendorf 5414C microcentrifuge for 30min at 4° C. The supernatant was decanted carefully, the DNA pellet wasrinsed with cold 75% (v/v) ethanol and centrifuged briefly. The pelletwas dried in a vacuum desiccator for 10 min and the DNA was finallydissolved in 20 μl of TE and stored at 4° C.

Ligation of PCR-Amplified Niale-Associated Fragments into Plasmid Vector

Fragments resulting from PCR with RAPD primers OPAC.06. OPAD.11. OPAM.01and OPAM.07 were ligated into plasmid pGEM-T (a linearised derivative ofpGEM-3) using the pGEM-T vector cloning system (Promega) according tothe supplier's instructions.

Fragments resulting from PCR with RAPD primer OPAI.05 were cloned byblunt-end ligation into the plasmid vector pGem-3Z (Promega). The vectorwas linearised by digestion with restriction endonuclease SmaI (NewEngland BioLabs) in NEBuffer 4 (New England BioLabs) then treated withcalf alkaline phosphatase (New England BioLabs). The digested plasmidDNA was purified by electrophoresis in 1% (w/v) LMP agarose (Sigma) in0.5×TBE buffer. The gel was stained with crystal violet (58), a minimalportion of gel containing the linear plasmid was excised and DNA wasrecovered as described above.

The gel-purified OPAI.05 male-associated RAPD material was treated withT4 DNA polymerase (New England BioLabs) according to the supplier'sinstructions then heated at 65° C. for 15 min. The cooled sample wasthen treated with T4 polynucleotide kinase (New England BioLabs)according to the supplier's instructions, then again heated at 65° C.for 15 min. The gel-purified linear vector (approx. 10 ng) and PCRfragments (approx. 5 ng) were ligated with 3 Weiss units of T4 DNAligase (Promega) in 50 μl of Promega DNA ligase buffer (30 mM Tris-HCl,pH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5mM ATP) at 4° C. for 14-16 h.

Transformation with Recombinant Plasmids

Single colonies of Escherichia coli strain DH5a (fragments from RAPD PCRwith primers OPAC.06, OPAD.11, OPAM.01 and OPAM.07) or strain XL1-Blue(fragments from RAPD PCR with primer OPAI.05) were inoculated into 200ml of LB broth and grown in a shaking incubator at 37° C. to an opticalabsorbance of approx. 0.3 at 550 nm (3-4 h). The cells were collected bycentrifugation at 3000 rpm for 5 min at 4° C. in an Eppendorf 5414Cmicrocentrifuge, resuspended in 30 ml of cold 0.1M MgCl₂ and placed onice for 20 min. The cells were collected by centrifugalion as before andthe pellet suspended in 1 ml of cold 0.1M CaCl2. Glycerol was added to15% (v/v) and the competent cells were stored at −70° C.

For transformation, 50 μl of competent cells was thawed and mixed with 5μl of ligation reaction, placed on ice for 20 min. heat-shocked at 42°C. for 45 sec then returned to ice for 5 min. The transformed cells wereallowed to recover by incubation at 37° C. for 1 h in 500 μl of SOCmedium (2% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract, 10 mMNaCl, 2.5 mM KCl, 10mM MgCl₂, 10 mM MgSO₄, 20 mM glucose) and were thenplated onto LB agar containing ampicillin (100 μg/ml), X-gal (25 μg/ml)and IPTG (10 μM) for overnight culture at 37° C. Transformationefficiency was 2×10⁷ cfu/μg plasmid (with ccc pGEM-T).

Colony Screening by PCR

White colonies were selected and incubated overnight in 500 μl of LBbroth. Inserts in recombinant plasmids of the cloned cells were analysedby PCR amplification from primer sites flanking the cloning site. One 1μl of the cell suspension was mixed with 2.7 μM each of the SP6(5′-ATTTAGGTGACACTATAGAATAC-3′ (SEQ ID NO:28)) and T7(5′-ATTATGCTGAGTGATATCCCGCT-3′ (SEQ ID NO:29)) primers (both fromBresatec Custom Oligos), 200 μM of each of the four dNTPs, 1.5 mM MgCl₂,100 mM tris-HCl, pH 8.3, 500 mM KCl and 1 unit of Taq DNA polymerase(Boehringer Mannheim) in a final volume of 25 μl.

Reactions were cycled in a Corbett Research PC-960 Air-CooledThermocycler with an initial step at 94° C. for 2 min followed by 35cycles of 94° C. for 20 sec, 50° C. for 20 sec and 72° C. for 30 sec; oncompletion of cycling the samples were heated at 72° C. for 2 min.

Colony Screening by Dot Blot Hybridization

Colonies that were found by PCR to contain a recombinant insert ofappropriate size (i.e. appropriate to the size of the male-associatedfragment generated from genomic DNA by RAPD PCR) were labelled byincorporating 8 μM digoxigenin-11-dUTP (DIG-dUTP: Boehringer Mannheim)in colony PCR reactions, as described above.

A replicate dilution series of male and female horse genomic DNA samples(1 μg, 250 ng, 100 ng and 10 ng of each) were denatured in 0.2 ml of0.4M NaOH, 10 mM EDTA and heated at 100° C. for 10 min. The samples wereapplied to positively-charged nylon membrane (Boehringer Mannheim; 59)with a Vacuum Blot Manifold (Gibco-BRL). Each well was washed with 500μl of 0.4M NaOH and the membrane was neutralised by 3×10 min washes in2×SSC.

DNA hybridizations were performed according to the DIG System User'sGuide for Filter Hybridization (Boehringer Mannheim). Membranes wereprehybridized at 50° C. for at least 2 h in 10 ml of DIG Easy Hybhybridization buffer (Boehringer Mannheim, cat. no. 1603558) in glasshybridization bottles (Hybaid) placed in a Eurotherm 91E RotatingHybridization Incubator (Model 310; Robbins Scientific).

DIG-labelled probes were prepared as described above from the inserts ofrecombinant plasmids, using the SP6 and T7 primers. Each was added to 4ml of DIG Easy Hyb solution (Boehringer Mannheim) at a concentration of50-100 ng/ml and denatured at 68° C. for 10 min. The prehybridizationsolution was replaced by the probe solution and hybridization wasconducted in the rotating incubator at 50° C. for 14-16 h.

The membrane was then removed and washed for 3×10 min at low stringency(2×SSC, 0.1% (w/v) SDS, 25° C.) followed by 2×10 min at high stringency(0.2×SSC, 0.1% (w/v) SDS, 68° C.). The washed membrane wasrotary-incubated for 1 h in 2×blocking solution (Boehringer Mannheim,cat. no. 1585762) containing 1×maleic acid buffer (Boehringer Mannheim,cat. no.1585762).

Anti-DIG antibody labelled with alkaline phosphatase (BoehringerMannheim, cat. no. 1093274) was added to the blocking solution at aconcentration of 0.075 units/ml and rotary incubation continued for afurther 30 min.

The membrane was then washed for 2×15 min in 1×wash buffer (BoehringerMannheim, cat. no. 1585762) and transferred to 1×detection buffer(Boehringer Mannheim. cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat. no.1685627) was diluted 1:100 in detection buffer and 1 ml was added per150 cm² of membrane. The substrate solution was spread evenly betweenclear transparency sheets and the signal was detected at roomtemperature using X-ray film (AGFA Eurix RP1) with intensifying screens(Dupont Quanta III-T).

Differential intensity of hybridization to male and female DNA samplesindicated probes derived from clones containing a male-associatedfragment from RAPD PCR with each of primers OPAC.06, OPAD.11, OPAI.05and OPAM.07 (data not shown). Attempts to recover a clonedmale-associated fragment from RAPD PCR with primer OPAM.01 wereunsuccessful.

Example 2

Sequence Analysis of Cloned Male-Associated Fragments

DNA sequencing was performed using dideoxy sequencing chemistryutilising the ABI PRISM™ Dye Terminator Cycle-Sequencing-Ready ReactionKit (ABI Perkin-Elmer) with AmpliTaq DNA polymerase, according to themanufacturer's instructions (ABI Perkin-Elmer). Products of sequencingreactions were analysed according to the manufacturer's instructions onan ABI A373 sequencer at the University of Queensland DNA Sequence andAnalysis Facility.

The sequence of cloned inserts of recombinant plasmids, derived fromrecovered products of RAPD PCR with each of primers OPAC.06, OPAD.11,OPAI.05 and OPAM.07, that hybridized differentially with male DNA on dotblots are shown in SEQ ID NOS: 1, 2, 3 and 4, respectively. Theseinserts are known henceforth as EY.AC6, EY.AD11, EY.AI5 and EY.AM7,respectively.

Hybridization Analysis of Cloned Male-Associated Fragments

Samples of genomic DNA (2.5 μg) from nine male and nine female horseswere digested with 5 units of Sau3AI (New England BioLabs) in NEBuffer(New England BioLabs: 100 mM NaCl, 10 mM bis-tris-propane-HCl, pH 7.0 at25° C., 10 mM MgCl₂, 1 mM, dithiothreitol) and 0.1 mg/ml BSA in a finalvolume of 25 μl.

The digested samples, together with a DIG-labelled DNA molecular weightmarker mix (Boehringer Mannheim. cat. no. 1218603), were electrophoresedin 1% (w/v) agarose at 70V for 3 h in 0.5×TBE. Resolved fragments werecapillary-transferred overnight in 0.4M NaOH to a positively-chargednylon membrane (Boehringer Mannheim; 30). Following transfer, themembrane was neutralised with 3×10 min washes in 2×SSC. Allhybridizations were performed according to the DIG System User's Guidefor Filter Hybridization (Boehringer Mannheim).

Membranes were prehybridized at 50° C. for at least 2 h in 10 ml of DIGEasy Hyb hybridization buffer (Boehringer Mannheim. cat. no. 1603558) inglass hybridization bottles (Hybaid) placed in a Eurotherm 91E RotatingHybridization Incubator (Model 310: Robbins Scientific).

DIG-labelled DNA probes were prepared as described above from the fourrecombinant plasmids containing inserts EY.AC6, EY.AD11, EY.AI5 andEY.AM7. Each was added to 4 ml of DIG Easy Hyb solution (BoehringerMannheim) at a concentration of 50-100 ng/ml and denatured at 68° C. for10 min. The prehybridization solution was replaced by the probe solutionand hybridization was conducted in the rotating incubator at 50° C. for14-16 h.

The membrane was then removed and washed for 3×10 min at low stringency(2×SSC, 0.1% (w/v) SDS, 25° C.) followed by 2×10 min at high stringency(0.2×SSC, 0.1% (w/v) SDS, 68° C.). The washed membrane wasrotary-incubated for 1 h in 2×blocking solution (Boehringer Mannheim,cat. no. 1585762) containing 1×maleic acid buffer (Boehringer Mannheim,cat. no. 1585762).

Anti-DIG antibody labelled with alkaline phosphatase (BoehringerMannheim, cat. no. 1093274) was added to the blocking solution at aconcentration of 0.075 units/ml and rotary incubation continued for afurther 30 min.

The membrane was then washed for 2×15 min in 1×wash buffer (BoehringerMannheim, cat. no. 1585762) and transferred to 1×detection buffer(Boehringer Mannheim, cat. no. 1585762) for 5 min. The chemiluminescentsubstrate CDP-Star (Boehringer Mannheim, cat. no. 1685627) was diluted1:100 in detection buffer and 1 ml was added per 150 cm² of membrane.The substrate solution was spread evenly between clear transparencysheets and the signal was detected at room temperature using X-ray film(AGFA Eurix RP1) with intensifying screens (Dupont Quanta III-T).

The male-differential hybridization pattern using EY.AI5 indicated thatthis sequence is present in multiple copies in the DNA of all malehorses surveyed. A homologous sequence is present in the female genomebut is much less abundant, where the relative intensity and pattern ofhybridization are suggestive of just one or a few copies.

In order to confirm that the cloned fragment EY.AI5 represents acanonical genomic repeated element, a DIG-labelled probe was prepared bydirect PCR of male genomic DNA using primers EQYL2:5′-AGCGGAGAAAGGAATCTCTGG-3′ (SEQ ID NO: 12) and EQYR4:5′-TTCGTCCTCTATGTTGAAATCAG-3′ (SEQ ID NO: 14) derived from the sequenceof EY.AI5 (nts 6-26 and the reverse complement of nts 173-195.respectively, in SEQ ID NO: 3; both primers provided by Bresatec CustomOligos).

The hybridization patterns with both probes are similar, although thedirect genomic probe appeared to hybridize relatively more strongly withfragments smaller than 900 bp in both male and female DNA, suggestingthat genomic representatives of the repeat include sequences that arenot part of the cloned EY.AI5 fragment.

The four cloned sequences EY.AC6, EY.AD11, EY.AI5 and EY.AM7 weresubsequently DIG-labelled and hybridized with Southern blots of male andfemale genomic DNA that had been digested with nine differentrestriction enzymes. All showed male-specific hybridization patterns butalso hybridized with female DNA, albeit to a significantly lesserextent.

These data demonstrate that each of the four sequences is repeated manytimes in the male genome and hence, by comparison with hybridization tofemale DNA, on the Y chromosome.

The striking similarity of hybridization patterns with all four probesto fragments cut by restriction enzymes having a six-base recognitionsequence (KpnI, EcoRI, HindIII, BamHI) implies that all four clonedfragments are components of a single long-range tandem repeat in theequine Y chromosome. Sequence analysis of the four cloned fragmentsrevealed overlap between EY.AC6 and EY.AM7 (SEQ ID NOS: 1 and 4),consistent with this interpretation.

Of the four cloned sequences, EY.AI5 showed the greatest quantitativedifference between male and female DNA. Restriction patterns suggestthat it has a basic repeat unit in the genome of approximately 230 bp(TaqI and RsaI digests), consistent with the length of the sequencedisolate (SEQ ID NO: 3).

Using the conditions described above, the cloned sequence EY.AI5 wasDIG-labelled and hybridized with Southern blots of male and femalegenomic DNA that had been isolated from a variety of horse breeds anddigested with Sau3AI (FIG. 1). Hybridization patterns were similar forall breeds examined, including the subspecies known as Przewalski'shorse. This confirms the sex-differential occurrence of EY.AI5 sequencesthroughout the species Equus caballus.

Example 3

Conceptual Basis for Discriminatory PCR-Based Sexing Assay

Each of the four male-associated DNA sequences is clearly present in theequine Y chromosome since each shows a male-specific hybridizationpattern, but none is unique to the male genome. Considering the fourcandidates as targets for a diagnostic test for the equine Y chromosome,the EY.AI5 fragment appears to offer most promise in that it shows thegreatest differential between abundance on the Y chromosome andelsewhere. Accordingly, further studies focused on this sequence in anattempt to develop PCR conditions that would provide absolutediscrimination between male and female equine DNA by utilising potentialdifferences between the sequence on the Y chromosome and itshomologue(s) elsewhere in the genome.

The fact that the EY.AI5 sequence is repeated on the Y chromosomeimplies that it is not represented by a single, definable sequence;repeated DNA elements invariably show sequence heterogeneity (e.g. 60).Cloning of PCR-amplified sequences yields single, specificrepresentatives that, in addition to intrinsic sequence variations, mayadditionally contain errors due to incidental in vitro and in vivomutagenesis.

Furthermore, EY.AI5-primed sequence(s) present in female genomes must beanalysed to allow identification of possible sequence differencesbetween it/them and Y-chromosomal representatives.

Analysis of EY.AI5 Sequences in Male and Female Genomic DNA

For the above reasons, samples of genomic DNA from individual male andfemale horses were amplified by PCR from a pair of primers specific tothe sequence EY.AI5. Primers EQYL1: 5′-GTCGTAGCGGAGAAAGGAATC-3′ (SEQ IDNO: 15) and EQYR1: 5′-AGCGGACTGTTCCGTTTCGG-3′ (SEQ ID NO: 16) derivedfrom the sequence of EY.AI5 (nts 1-21 and the reverse complement of nts206-225, respectively, in SEQ ID NO: 3) were used to amplify genomic DNAtargets from a male and female horse (both primers provided by BresatecCustom Oligos). The products were sequenced directly from these primers,without cloning, to allow sequence analysis of the bulk population ofrepeated elements.

The sequence data (FIG. 2) show minor variations between the individual(cloned) representative EY.AI5 and the bulk sequence population in themale.

Two regions in fragments derived directly from the male genome differfrom the equivalent regions in female-derived fragments (nts 1-30 andnts 162-220). These regions of sequence divergence were chosen as theannealing targets for PCR primers designed to discriminate betweenEY.AI5 sequences in male and female genomic DNA.

Example 4

Development of PCR-Based Equine Sexing Assay

A primer pair was derived from the sequence data of FIG. 2 for specificdetection of equine Y-chromosomal DNA. These primers are EQYL2:5′-AGCGGAGAAAGGAATCTCTGG3′ (SEQ ID NO: 12) and EQYR5:5′-TACCTAGCGCTTCGTCCTCTAT-3′ (SEQ ID NO: 13), derived from nts 6-26 andthe reverse complement of nts 184-205, respectively, of the male genomicDNA sequence shown in FIG. 2 (underlined). These two regions exhibitsignificant sequence differences between male and female genomes.

Amplification of equine genomic DNA samples (15 pg to 2 ng) from theseprimers (both primers provided by Bresatec Custom Oligos) yielded aproduct of approximately 200 bp from male DNA samples and no detectableproduct from female DNA samples (data not shown). Analysis of genomicDNA samples from ten unrelated horses (data not shown) confirmed thatPCR amplification from these primers provides an accurate means ofdetecting the presence of Y-chromosomal DNA sequences.

In a diagnostic assay for genetic sex, no detectable product of PCRamplification from male-specific primers may result not only from afemale sample but from PCR failure or loss of sample. The possibility offalse negative results must be minimised. For this reason, a duplex PCRassay was developed in which a 121 bp (approximately) fragment of adispersed autosomal repeated sequence (SINE; 61) was amplifiedsimultaneously with the Y-specific target.

Primers used to amplify the SINE element were EQSIN8:5′-GCCCAGTGTTTCGTTGGTTCG-3′ (SEQ ID NO: 17) and EQSIN9:5′-CATAGTTGTATATTCTTCGTTGTGG-3′ (SEQ ID NO: 18), derived from nts 53-72and the reverse complement of nts 148-172, respectively, of the ERE-1SINE sequence family (61).

Duplex PCR amplifications were mutually optimised by inclusion of acommon ml3 sequence at the 5′-termini of all four primers (62.63). Thetwo primer pairs used for duplex equine sexing by PCR were:

sexing primers:

mEQYL2 5′-GCGGTCCCAAAAGGGTCAGTAGCGGAGAAAGGAATCTCTGG-3′ (SEQ ID NO: 19)

mEQYR5 5′-GCGGTCCCAAAAGGGTCAGTTACCTAGCGCTTCGTCCTCTAT-3′ (SEQ ID NO: 20)

control primers:

mEQSIN8 5′-GCGGTCCCAAAAGGGTCAGTGCCCAGTGTTTCGTTGGTTCG-3′ (SEQ ID NO: 21)

mEQSIN9 5′-GCGGTCCCAAAAGGGTCAGTCATAGTTGTATATTCTTCGTTGTGG-3′ (SEQ ID NO:22)

Duplex PCR reactions for internally-controlled assay of equine geneticsex were conducted in plastic capillary tubes (for use with the CorbettResearch FT'S-1 thermal cycler) containing 50 mM KCl, 10 mM Tris-HCl, pH8.3, 0.001%(w/v) gelatin, 2 mM MgCl₂, 100 μM dATP, 100 μM dCTP, 100 μMdGTP, 100 μM dTTP, 0.2 μM mEQSIN8, 0.2 μM mEQSIN9, 0.45 μM mEQYL2, 0.45μM mEQYR5 (all four primers provided by Bresatec Custom Oligos) and 0.5units of AmpliTaq (Perkin-Elmer) in a total volume of 10 μl.

Samples were placed in a Corbett Research FTS-1 capillary thermal cyclerand subjected to a heating program of 94° C. for 30 sec, 69° C. for 30sec and 72° C. for 30 sec for a total of six cycles, followed by anadditional 20 cycles of 94° C. for 30 sec and 72° C. for 30 sec, thenfinally held at 25° C. pending analysis by agarose gel electrophoresis.

The products were electrophoresed in 2% (w/v) agarose gel in TAE buffer(Boehringer Mannheim) at 100V for approximately 30 min, then stainedwith ethidium bromide and visualised under uv irradiation. Duplex PCRamplification of equine genomic DNA samples resulted in a single visiblefragment of approximately 160 bp from female horse DNA, resulting fromamplification of SINE elements. Male DNA gave rise to a similar band andan additional band at approximately 240 bp, resulting from amplificationof Y-chromosomal EY.AI5 elements. No product was seen in the absence ofDNA.

The duplex PCR sexing assay described above is clearly able to identifyand discriminate between male and female DNA samples, from 5 ng to aslittle as 20 pg (approximately equivalent to the amount of DNA in threecells).

Example 5

Application of Duplex PCR Sexing Assay to Horses of Various Breeds

Samples of DNA isolated from male and female horses of various breedswere analysed by duplex PCR as described above.

Duplex PCR with the sexing and control primers was able to identify anddiscriminate between DNA samples from male and female horses of allbreeds, with similar results for all breeds including the subspeciesknown as Przewalski's horse.

Example 6

Application of Duplex PCR Sexing Assay to Whole Blood Cells

The preceding examples illustrate successful application of thedescribed duplex PCR sexing assay to small DNA samples. For ease ofutility it is desirable to conduct the assay on small numbers of cellswithout the necessity to isolate DNA from them. White blood cells wereused to establish appropriate assay conditions.

Blood samples were withdrawn into Vacutainer® CPT™ tubes with sodiumcitrate (Becton Dickinson). Tubes were kept upright at room temperatureand processed within 2 hours of collection.

Each tube was centrifuged in a swinging bucket rotor (Sigma 3K18 with11133 rotor) at 1900 g for 30 min at 24° C. Approximately 60-70% of theclear plasma layer was removed then the remaining liquid above the gelmatrix, substantially free of red blood cells, was transferred into aclean tube. The sample was diluted to 10 ml with PBS and centrifuged at300×g for 15 min at 24° C. Supernatant was removed and the pelletresuspended gently in 10 ml of PBS and centrifuged under the sameconditions. The pellet was again suspended in 10 ml of PBS thencentrifuged at 100×g.

The cell pellet was resuspended in 250 μl of PBS and the suspensioncounted by haemocytometer to determine the concentration of nucleatedcells. The suspension was finally diluted in PBS to a concentration of10⁶ nucleated cells/ml.

Samples of cell suspensions from male and female horses were seriallydiluted in PBRS containing 50 mM DTT and appropriate dilutions weresubjected to two successive freeze/thaw cycles: tubes containing thesamples were initially floated on liquid nitrogen for 1-2 min untilfrozen, then transferred to a water bath at room temperature until thesuspension thawed. The tubes were placed in a boiling water bath for 15min then cooled on ice. Duplex PCR reactions were conducted in plasticcapillary tubes (for use with the Corbett Research FTS-1 thermal cycler)containing 50 mM KCl, 10 mM Tris-HCl, pH 8.3, 0.001%(w/v) gelatin, 2mMMgCl₂, 100 μM dATP, 100 μM dCTP, 100 μM dGTP, 100 μM dTTP, 0.2 μMmEQSIN8, 0.2 μM mEQSIN9, 0.45 μM mEQYL2, 0.45 μM mEQYR5, 0.5 units ofAmpliTaq and 2 μl of treated cell suspension in a total volume of 10 μl.

Samples were placed in a Corbett Research FTS-1 capillary thermal cyclerand subjected to a heating program of 94° C. for 30 sec. 69° C. for 30sec and 72° C. for 30 sec for a total of six cycles, followed by anadditional 21 cycles of 94° C. for 30 sec and 72° C. for 30 sec, thenfinally held at 25° C. pending analysis by agarose gel electrophoresis.

The products were electrophoresed in 2% (w/v) agarose gel in TAE buffer(Boehringer Mannheim) at 100V for approximately 30 min, then stainedwith ethidium bromide and visualised under uv irradiation.

As before, no bands were observed in the absence of equine DNA whereasbands are clearly visible with 20 pg of DNA from a female horse (singleband at approximately 160 bp) and a male horse (two bands, atapproximately 160 bp and 240 bp).

Samples of white blood cells containing approximately 5 cells to 100cells each yielded one (160 bp) or two (160 and 240 bp) bands,consistent with their origin from female or male horses, respectively.

Example 7

Application of Duplex PCR Sexing Assay to Equine Embryos

Eight embryos were recovered from five mares approximately eight daysafter fertilisation and each was immediately split into four or moresections depending on the size of the blastocyst (each sample containedan estimated maximum of 50 cells). Splitting was performed bymicromanipulation (1,2) in 50 μl of PBS. Each section of the blastocystwas collected with 2 μl of 4% (w/v) BSA (Miles Pentex crystalline, cat.no. 81-001-4; 1,2) and transferred into 7.5 μl of deionised water. Thesections were stored frozen at −20° C.

The thawed suspension of each embryo section, containing approximately15-50 cells, was made 20 mM in DTT and dispensed randomly into tubesnumbered from 8 to 39. The 32 samples were from this stage processed‘blind’ by a second individual who had not been involved with embryocollection, splitting or sample preparation.

Each sample was subjected to two successive freeze/thaw cycles. For eachcycle the tubes containing the samples were floated on liquid nitrogenfor 1-2 min until frozen, then transferred to a water bath at roomtemperature until the suspension thawed. The tubes were finally placedin a boiling water bath for 15 min then cooled on ice.

Duplex PCR reactions were conducted in plastic capillary tubes (for usewith the Corbett Research FTS-1 thermal cycler) containing 50 mM KCl, 10mM Tris-HCl, pH 8.3, 0.001%(w/v) gelatin, 2 mM MgCl₂, 100 μM dATP, 100μM dCTP, 100 μM dGTP, 100 μM dTTP, 0.2 μM mEQSIN8, 0.2 μM mEQSIN9, 0.6μM mEQYL2, 0.6 μM mEQYR5, 0.5 units of AmpliTaq and 9.5 μl of embryocell suspension in a total volume of 20 μl.

Samples were placed in a Corbett Research FTS-1 capillary thermal cyclerand subjected to a heating program of 94° C. for 30 sec, 69° C. for 30sec and 72° C. for 30 sec for a total of six cycles, followed by anadditional 21 cycles of 94° C. for 30 sec and 72° C. for 30 sec, thenfinally held at 25° C. pending analysis by agarose gel electrophoresis.

The products were electrophoresed in 2% (w/v) agarose gel in TAE buffer(Boehringer Mannheim) at 100V for approximately 30 min, then stainedwith ethidium bromide and visualised under uv irradiation.

No bands are observed in the absence of equine DNA whereas bands areclearly visible with 20 pg of DNA from female DNA (single band atapproximately 160 bp) and male DNA (two bands, at approximately 160 bpand 240 bp). Bands are clearly visible with both 125 and 5(approximately) white blood cells from a female horse (single band atapproximately 160 bp) and a male horse (two bands, at approximately 160bp and 240 bp).

Products resulting from assay of embryo sections showed relatively weaksignals which were variable in intensity; this was found subsequently toresult from sub-optimal PCR conditions due to a pH shift caused by theBSA used in the collection of embryo sections.

Two individuals, who had not been involved in earlier stages of theanalysis, independently called the sex of each embryo section from theassay results. The calls of both individuals were in complete agreementand are shown in Table 1.

Table 1 shows the analysis of sex of embryo biopsies by duplex PCR.Embryos were recovered from mares approximately eight days afterfertilisation, cut into four or more sections and the sections frozen at−20° C. The thawed sections, each containing approximately 15-50 cells,were dispensed randomly into tubes numbered from 8 to 39 and the 32samples were analysed ‘blind’ by duplex PCR, as described in the text.At the conclusion of assay, two further individuals independently calledthe sex of each assay result. The calls of both individuals were incomplete agreement. F is female diagnosis, M is male diagnosis and NR isno result. The data have been rearranged for clarity.

For every embryo, all four sections from the same embryo were called asthe same sex, with the exception of sample 34 which yielded no result(neither male-specific band nor control band was visible, confirming thevalue of including primers for an internal control). The probability ofsuch a result arising by chance is <10⁻⁷.

These data provide statistical validation of the duplex PCR sexing assayfor embryo sections.

TABLE 1 Embryo no. Estimated no. cells Sample ID Sex called 1 50 12 F 150 39 F 1 50 20 F 1 50 33 F 2 45 10 M 2 45 13 M 2 45 30 M 2 35 24 M 3 3031 F 3 35 17 F 3 45 37 F 3 35 19 F 4 50 36 M 4 50  8 M 4 50 21 M 4 50 38M 5 50 27 F 5 30 16 F 5 50 35 F 5 50 29 F 6 50 25 M 6 50 14 M 6 50 23 M6 50 11 M 7 50 32 M 7 50 28 M 7 50  9 M 7 50 22 M 8 15 26 M 8 25 18 M 840 15 M 8 40 34 NR

Example 8

Identification of Long-Range Repeat in the Equine Y Chromosome

To investigate the inter-relation, repetition, conservation and genomicenvironment of the four described sequence elements associated with theequine Y chromosome, these elements were used as probes to identifyrecombinant bacteriophage in an equine genomic library.

Equine Genomic Library

A male horse genomic DNA library in the Lambda Fix® II vector wasobtained from Stratagene (cat. no. 946701). The estimated titre of thelibrary after a single round of amplification was 2.0×10⁹ plaque formingunits (pfu)/ml. Phage and host bacteria were cultured according tomethods detailed in the instruction manual provided with the library(Stratagene).

Screening of Equine Genomic Library with EY.AI5 Probe

For the first round of screening a total of 25,000 to 30,000 plaquesgrown in host E. coli XL1-Blue NIRA (P2) were present on each 150 mmplate of growth medium. Duplicate plaque lifts were made from each plateand DNA was bound to uncharged Nylon Membranes for Colony and PlaqueHybridization (132 mm diameter) purchased from Boehringer Mannheim (cat.no. 1699083) according to protocols outlined in the DIG System User'sGuide for Filter Hybridization (Boehringer Mannheim). After uvcross-linking (Bio-Rad GS Gene Linker), membranes were prehybridized at42° C. for at least 2 h in 10 ml of DIG Easy Hyb hybridization buffer(Boehringer Mannheim, cat. no. 1603558) in glass hybridization bottles(Hybaid) placed in a Eurotherm 91E Rotating Hybridization Incubator(Model 310: Robbins Scientific). A DIG-labelled EY.AI5 probe and acontrol probe for a 121 bp (approximately) fragment of a dispersedautosomal repeated sequence (SINE: 61) were prepared as described abovefrom the inserts of recombinant plasmids, using the SP6 and T7 primers.Each was added to 10 ml of DIG Easy Hyb solution (Boehringer Mannheim)at a concentration of 25-50 ng/ml and denatured at 68° C. for 10 min.The prehybridization solution was replaced by the probe solution andhybridization was conducted in the rotating incubator at 42° C. for14-16 h.

The membrane was then removed and washed for 3×10 min at low stringency(2×SSC, 0.1% (w/v) SDS, 25° C.) followed by 2×10 min at high stringency(0.2×SSC, 0.1% (w/v) SDS, 68° C.). The washed membrane wasrotary-incubated for 1 h in 2×blocking solution (Boehringer Mannheim,cat. no. 1585762) containing 1×maleic acid buffer (Boehringer Mannheim,cat. no. 1585762).

Anti-DIG antibody labelled with alkaline phosphatase (BoehringerMannheim, cat. no. 1093274) was added to the blocking solution at aconcentration of 0.075 units/ml and rotary incubation continued for afurther 30 min.

The membrane was then washed for 2×15 min in 1×wash buffer (BoehringerMannheim, cat. no. 1585762) and transferred to 1×detection buffer(Boehringer Mannheim, cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat. no.1685627) was diluted 1:100 in detection buffer and 1 ml was added per150 cm² of membrane. The substrate solution was spread evenly betweenclear transparency sheets and the signal was detected at roomtemperature using X-ray film (AGFA Eurix RP1) with intensifying screens(Dupont Quanta III-T).

Fifty plaques of the 100 (approximately) plaques that gave positivesignals in duplicate were selected from the 300,000 that were screenedand each was removed in a small agar plug; the plugs were stored in SMTbuffer at 4° C.

A second round of screening was conducted at reduced plaque density(approx. 3.500 pfu) for 20 of the positives. Methods for plaque liftsand hybridizations were identical to those used in first roundscreening. Sixteen independent clones positive for EY.AI5 probe (onduplicate filters) were selected for further investigation. These were:31.1, 31.2, 31.3. 31.4, 31.5 (originating from five positive plugsselected from plate number 31 in the first screening round). 32.3. 33.1.33.2, 33.4, 34.1, 34.2, 34.3. 34.5, 36.1, 36.2 and 36.3 (similarlyselected from plates 32, 33, 34 and 36. respectively).

Isolation of DNA from Recombinant Phage

DNA was isolated from five positive clones using host strain E. coliXL1-Blue NIRA growing at 37° C. overnight in 50 ml LB mediumsupplemented with 0.3% (v/v) glycerol and 10 mM MgSO4 (64). DNA (200 μg)was isolated using anion-exchange resin under appropriate salt and pHconditions (QIAGEN Lambda Maxi Kit; cat. no. 12562) in accordance withthe methods supplied by the manufacturers.

Southern Hybridization of Phage Inserts with EY.AI5 and EY.AD11 Probes

Twenty μg of recombinant phage DNA was incubated overnight with 40 unitsof restriction enzyme EcoRI or HindIII. All restriction digests werecarried out with enzymes supplied by Boehringer Mannheim or New EnglandBiolabs in buffers as supplied by the manufacturers and in accordancewith their instructions. Aliquots of 10 μg of digested DNA, togetherwith a DIG-labelled DNA molecular weight marker mix (BoehringerMannheim, cat. no. 1218603), were electrophoresed in 1% (w/v) agarose at80V for 5 h in 0.5×TAE. Resolved fragments were capillary-transferredovernight in 0.4M NaOH to a positively-charged nylon membrane(Boehringer Mannheim; 30). Following transfer, the membrane wasneutralised with 3×10 min washes in 2×SSC then DNA was uv cross-linkedto the membrane (Bio-Rad GS Gene Linker).

All hybridizations were performed according to the DIG System User'sGuide for Filter Hybridization (Boehringer Mannheim). Membranes wereprehybridized at 42° C. for at least 2 h in 10 ml of DIG Easy Hybhybridization buffer (Boehringer Mannheim. cat. no. 1603558) in glasshybridization bottles (Hybaid) placed in a Eurotherm 91E RotatingHybridization Incubator (Model 310: Robbins Scientific).

DIG-labelled DNA probes were prepared as described above from the tworecombinant plasmids containing inserts EY.AD11 and EY.AI5 (Example 2).Probe was added to 10 ml of DIG Easy Hyb solution (Boehringer Mannheim)at a concentration of 25-50 ng/ml and denatured at 68° C. for 10 min.The prehybridization solution was replaced by the probe solution andhybridization was conducted in the rotating incubator at 42° C. for14-16 h.

The membrane was then removed and washed for 3×10 min at low stringency(2×SSC, 0.1% (w/v) SDS, 25° C.) followed by 2×10 min at high stringency(0.2×SSC, 0.1% (w/v) SDS, 68° C.). The washed membrane wasrotary-incubated for 1 h in 2×blocking solution (Boehringer Mannheim.cat. no. 1585762) containing 1×maleic acid buffer (Boehringer Mannheim.cat. no. 1585762).

Anti-DIG antibody labelled with alkaline phosphatase (BoehringerMannheim, cat. no. 1093274) was added to the blocking solution at aconcentration of 0.075 units/ml and rotary incubation continued for afurther 30 min.

The membrane was then washed for 2×15 min in 1×wash buffer (BoehringerMannheim, cat. no. 1585762) and transferred to 1×detection buffer(Boehringer Mannheim, cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat. no.1685627) was diluted 1:100 in detection buffer and 1 ml was added per150 cm² of membrane. The substrate solution was spread evenly betweenclear transparency sheets and the signal was detected at roomtemperature using X-ray film (AGFA Eurix RPI) with intensifying screens(Dupont Quanta III-T).

Following detection of the positive signals for EY.AI5 probe (FIG. 3a),the probe was stripped from the membrane by treatment with 0.1% (w/v)SDS in 0.2M NaOH at 68° C. for 20 min. The membrane was then hybridizedusing the methods detailed above with the probe for the EY.AD11 sequence(FIG. 3b). Most restriction fragments positive for EY.AI5 were alsopositive for EY.AD11. Phage 31.1 failed to exhibit hybridization witheither probe.

Phage restriction fragments were selected for sequence analysis based ontheir hybridization to both probes. The fragments were alike in size togenomic repeated elements identified by using the same two probes forSouthern analysis of equine genomic DNA digested with EcoRI and HindIII.Fragments selected for sequencing were: a 3.3 kb HindIII fragment fromphage 33.1 (similar in size to a HindIII fragment in both male andfemale genomic DNA at an intensity ratio of approximately 2:1); a 4.4 kbHindIII fragment from phage 36.1 (similar in size to a HindIII fragmentin both male and female genomic DNA at an intensity ratio ofapproximately 20:1); a 4.7 kb EcoRI fragment from phage 32.3 (similar insize to a genomic EcoRI band which hybridizes to both probes at lowintensity in the male and at a barely detectable level in the female);and a 6.0 kb EcoRI fragment from phage 36.1 (present in male genomic DNAbut not detected in the female).

Restriction Mapping of Recombinant Phage Inserts

Inserts of equine genomic DNA flanked by T3 and T7 promoter sites wereexcised from the Lambda Fix® II vector by digestion with NotI. Thiscassette (1 μg) was subjected to partial digestion with either EcoRI(for page 32.3 and 36.1) or HindIII (for phage 33.1 and 36.1). Thepartial digestion fragments, together with fragments of 1 DNA digestedwith HindIII and EcoRI as size markers (Boehringer Mannheim; cat. no.528552), were electrophoresed through a 1% (w/v) agarose gel at 80V for5 h in 0.5×TAE with ethidium bromide; the gel was then overlaid with ascale ruler and photographed with uv transillumination (302 nm).

The resolved fragments were transferred to an uncharged membrane(Hybond-N, Amersham cat. no. RPN303N) with 20×SSC following depurinationin 0.25M HCl, denaturation in 0.5M NaOH, 1.5M NaCl and neutralization in1M Tris (pH 7.5), 1.5M NaCl (65). The membranes were probed successivelywith biotinylated oligonucleotide probes for the T3 and T7 promotersequences, respectively (New England Biolabs cat. nos. 1227-BT and1223-BT); membranes were stripped before the second hybridization bytreatment with 0.1% (w/v) SDS in 0.2M NaOH at 68° C. for 20 min.Prehybridization was done in glass hybridization bottles (Hybaid) placedin a Eurothermi 91E Rotating Hybridization Incubator (Model 310: RobbinsScientific) in 10 ml phosphate buffered 7% (w/v) SDS solution with 1%(w/v) BSA and 0.5 mg/ml carrier DNA (28). Biotinylated oligonucleotideprobe was added to a final concentration of 10 ng/ml. Hybridizationtemperatures were 59° C. for the T7 probe and 49° C. for the T3 probe.Post-hybridization washes were in 5×SSC, 0.1% (w/v) SDS at 25° C. for 15min, then in 5×SSC. 0.1% (w/v) SDS at 60° C. for 15 min. membranes wereblocked with 10 ml of 1×Blocker (5.0% (w/v) SDS, 125 mM NaCl. 25 mMsodium phosphate (pH 7.2)) at 25° C. for 15 min. Alkalinephosphatase-conjugated streptavidin (Boehringer Mannheim: cat no.1093266) was added to the blocking mixture to a concentration of 1unit/ml and the membranes were treated for a further 10 min.Post-treatment washes were in 1×Blocker for 10 min then in two changesof 0.1×Blocker for 15 min followed by detection with CDP-Star andautoradiography as outlined above.

Sizes of hybridizing fragments were estimated by comparison withpositions of control size markers run concurrently in the gel. Theresulting ladder of DNA fragments corresponded to the distance from theT3 or T7 promoter site, respectively, and successive restriction sites(analagous to the ladder generated from labelled primers with dideoxyDNA sequencing). Because the T3 and T7 promoter sites flank the two endsof the insert, complementary maps were obtained, allowing confirmationof the position of restriction sites.

Analysis of complete digestion products on an ethidium bromide-stainedgel provided additional information regarding distances separating alladjacent cleavage sites. Restriction maps of phage clones 32.3 (EcoRI),33.1 (HindIII) and 36.1 (HindIII and EcoRI) are shown in FIGS. 4a, 5 aand 6 a, respectively.

Subcloning of Restriction Fragments from Recombinant Phage

Recombinant phage DNA (7.5 μg) was digested with 20 units of EcoRI (forphage 32.2 and 36.1) or HindIII (for phage 33.1 and 36.1) for 6 h at 37°C. Restriction fragments were resolved by electrophoresis in 1% (w/v)agarose in 0.5×TAE buffer containing ethidium bromide. Bands werevisualized with uv transillumination (302 nm) and the selected fragmentswere excised. DNA was recovered using silica-gel membrane technology(QIAquick Gel Extraction Kit; QIAGEN cat. no. 28704) in accordance withthe manufacturer s instructions.

The cloning vector was phagemid pBluescript® II KS⁺ (Stratagene) whichhad been linearized by digestion with either HindIII or EcoRI thentreated with shrimp alkaline phosphatase (Boehringer Mannheim; cat. no.1758250). Vector DNA was purified as above by gel electrophoresis andthe QIAquick gel extraction technique. The linear vector (approx. 20 ng)and phage restriction fragments (approx. 20 ng) were ligated with 1Weiss unit of T4 DNA ligase (Boehringer Mannheim) in 10 μl of T4 DNAligase buffer, as supplied with the enzyme, at 15° C. for 16 h. Ligationreactions were used to transform 100 μl of competent E. coli DH5 acells.

Single colonies of E.coli strain DH5 a were inoculated into 250 ml of LBbroth and grown in a shaking incubator at 37° C. to an opticalabsorbance of approx. 0.5 at 550 nm. The cells were collected bycentrifugation at 3.000 rpm for 5 min at 4° C. in an Eppendorf 5414Cmicrocentrifuge. resuspended in 30 ml of cold 0.1M MgCl₂ and placed onice for 20 min. The cells were collected by centrifugation as before andthe pellet suspended in 1 ml of cold 0.1M CaCl2. Glycerol was added to15% (v/v) and the competent cells were stored at −70° C.

For transformation, a 100 μl aliquot of competent cells was thawed andmixed with 10 μl of ligation reaction, placed on ice for 30 min,heat-shocked at 42° C. for 2 min then returned to ice for 2 min. Thetransformed cells were allowed to recover by incubation at 37° C. for 1h in 900 μl of SOC medium (2% (w/v) bacto-tryptone, 0.5% (w/v)bacto-yeast extract, 10M NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO4, 20mM glucose) and were then plated onto LB agar containing ampicillin (100μg/ml) for overnight culture at 37° C. Eight or sixteen colonies wereselected from ligations of each vector/insert pair.

A volume of 3 ml of LB broth containing ampicillin (100 μg/ml) wasinoculated with each colony and bacterial suspensions were harvestedafter 16 h at 37° C. in a shaking incubator. Miniprep DNA was preparedfrom each suspension (Wizard™ Plus Minpreps, Promega cat no. A7500).Purified plasmid DNA was compared with uncut vector by gelelectrophoresis to identify clones with plasmids containing an insert ofappropriate size.

DNA from these clones was digested with either HindIII or EcoRI, asappropriate, and clones with excisable inserts of the correct size wereused for sequence analysis. Insert-containing plasmid clones were asfollows: 32.3E1 and 32.3E5 (pBluescript® II with 4.7 kb EcoRI fragmentsfrom recombinant phage 32.3); 33.1H2, 33.1H3, 33.1H4, 33.1H6, 33.1H7 and33.1H8 (pBluescript® II with 3.3 kb HindIII fragments from recombinantphage 33.1); 36.1H1, 36.1H2, 36.1H4, 36.1H6, 36.1H7, 36.1H8, 36.1H9 and36.1H10 (pBluescript® II with 4.4 kb HindIII fragments from recombinantphage 36.1): and 36.1E2 and 36.1E8 (pBluescrip® II with 6.0 kb EcoRIfragments from recombinant phage 36.1).

Single colonies of E.coli DH5a with plasmids 32.3E5, 33.1H2, 33.1H7,36.1H7, 36.1H1 and 36.1E2 were used to prepare bacterial suspensions in50 ml LB broth with ampicillin (100 μg/ml). Preparations of 100-200 μgof plasmid DNA were purified from these suspensions according tosupplier's instructions using a QIAGEN Plasmid Kit and QIAGEN-tip 100resin columns (QIAGEN; cat. no. 12144).

Sequence Analysis of Subcloned Phase Fragments Hybridizing to ProbesEY.AI5 and EY.AD11

DNA sequencing was performed using dideoxy sequencing chemistryutilising the ABI PRISM™ BigDye Terminator Cycle Sequencing ReadyReaction Kit (ABI Perkin-Elmer) with AmpliTaq DNA polymerase accordingto the manufacturer's instructions (ABI Perkin-Elmer). Where apparentsecondary DNA structure in insert 36.1H7 impeded the terminatorsequencing reaction, the BigDye Primer Cycle Sequencing Ready ReactionKit (T7) (ABI Perkin-Elmer) was used. Products of sequencing reactionswere analysed according to the manufacturer's instructions on an ABIA377 sequencer at the Australian Genome Research Facility located at TheUniversity of Queensland.

A total of 112 sequencing reactions with an average read length of 700bases were undertaken. Approximately 20 kb of novel equine genomic DNAsequences were recorded then analysed, firstly for homology to sequencesdescribed herein as EY.AC7, EY.AM17, EY.AD11 and EY.AI5, and secondlyfor homology to sequences available worldwide in GenBank (NationalCenter for Biotechnology Information, Maryland, USA [NCBI]) and similarDNA and protein databases.

Primers for T3 and T7 promoter sequences flanking inserts inpBluescript® II vector (Bresatec Custom Oligos) were used for theinitial sequencing, steps. Sequences were extended from their 3′extremity with 19-24-mer oligonucleotide primers (Bresatec CustomOligos) designed for this purpose from known sequence data, thenmatching this extended sequence to the primary data. Replicate,overlapping and complementary strand sequencing assured the accuracy ofthe final genomic DNA sequences. Computer software used to constructcontiguous DNA sequences was Sequencher™ 3.0 (Gene Codes Corporation)for Macintosh®.

Eight 4.4 kb HindIII fragments subcloned from phage 36.1 proved to beidentical, differing only in the orientation of the insert in thevector. This conclusion was based on 100% identity observed withinblocks of at least 400 bases of sequence. Application of this criterionestablished that the two 6.0 kb EcoRI fragments from phage 36.1 werealso identical. Two subcloned EcoRI fragments of 4.7 kb from phage 32.3were not identical.

Mapping and restriction analysis of phage clone 33.1 (see FIG. 5a)indicated the presence of two similar 3.3 kb HindIII fragments. Partialsequencing of six subcloned 3.3 kb inserts supported this view. Insertsin plasmids 33.1H7, 33.1H3, 33.1H4 and 33.1H6 were identical; inserts inplasmids 33.1H2 and 33.1H8 were identical to each other but exhibited88-90% similarity with the former group. Accordingly, inserts inplasmids 33.1H7 and 33.1H2 were sequenced independently.

Sequencing of phage DNA selected for hybridization with EY.AI5 andEY.AD11 sequences (Example 2) revealed that these and the sequencesEY.AC6 and EY.AM7 (Example 2) were components of a long range repeatunit in the equine Y chromosome. The structure of the repeat as it wasfound in equine genomic DNA inserts in phage 32.3, 33.1 (twice) and 36.1is shown in FIGS. 4b, 5 b and 6 b.

Plasmid 32.3E1 contains an insert, possibly part of a pseudogene, withlimited homology to open reading frames of a number of unrelated genes,as determined by a database nucleotide and protein translation search(GenBank BLAST 2.0; blastn and blastx programs; NCBI).

The repeat element identified in equine genomic DNA comprises sequenceswhich include the aforementioned repeats EY.AI5, EY.AD11, EY.AC6 andEY.AM7. In addition, EY.AI5 sequence featured as part of a 1500 bp(approximately) unit, hereafter referred to as EY.LINE, found to beapproximately 50% homologous at the protein level (GenBank BLAST 2.0;blastx search: NCBI) to regions of open reading frame 2 (ORF2) in amammalian long interspersed repeated element (LINE: GenBank accessionnos. U93574 (human) and AB012223 (dog)). These elements, whenfunctional, are 6.0 kb in size and contain a 5′ untranslated region(UTR) with an internal promoter, two open reading frames (ORF1 and ORF2)and a 3′ UTR that terminates in a polyA tail. ORF1 encodes a nucleicacid binding protein and ORF2 encodes a protein with endonuclease andreverse transcriptase activities. The EY-LINE sequence is located atnucleotides 990-2497 of SEQ ID NO: 8, 421-1920 of SEQ ID NO. 9, 421-1930of SEQ ID NO. 10. and 1502-2996 of SEQ ID NO. 11.

LINEs are highly repetitive DNA sequence elements capable ofretrotransposition that pervade mammalian genomes. Most are functionallyinactive due to truncations, rearrangements and nonsense mutations (67).LINEs are present in all mammals that have been studied (includingmarsupials) and are thought to be derived from a common ancestor. Whencloned elements from a particular species are compared there may bedifferences between individual sequences. Southern hybridizations ofrestricted genomic DNA give species-specific patterns when hybridizedwith a LINE probe (68).

The aforementioned four DNA sequences associated with the equine Ychromosome, when used as probes, detect this EY.LINE repeated element.This element is, in turn, a part of a larger repeated element of 3.5 kb(minimum length) that is evident from comparison of FIGS. 4b, 5 b and 6b.This explains the similarity between Southern hybridizations using thedifferent sequences as probes. That the EY.LINE described here isspecific to Equidae is illustrated by FIG. 1 where no hybridization ofEY.AI5 is detected to restricted genomic DNA of the camel. The malespecificity of EY. LINE in Equus spp. is illustrated clearly by the dataof FIG. 1.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

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29 1 432 DNA Equus caballus 1 ccagaacgga gcaggccttt ccaagttcgaggagaaagtt agaaacctac aagaaatcca 60 gagaaagcaa aactatccta cctggaaagggggaggagtc agacaggtct gagatggctc 120 tgaaactctg tgtacttgga ggttgccaggacaacttagg tatctgaggt tgtttatgac 180 atcacggtga ccatgttccc caagttggccgcatggttac agtctgagaa ttgccctggc 240 tggtctatta aaggaagaca tacccagaaatagctttgac aaacagaagt cgtgcacaga 300 aactagaaga gatcaaacag actctgattacaggcataag aaaggactcc ttgccaagaa 360 gcgagaagag agggacaagc actgagaggagaacattctc atctgttcaa gtctatgggg 420 attccgttct gg 432 2 600 DNA Equuscaballus 2 caatcgggtc cagaataacg acatacagct gtgggggctg aaagagattagagaacgtga 60 acttccaagg attgaaaatc acctcaaaag tcttactgat gctacagaagggtagaccat 120 tccaaataca tgaagaggaa cactaccaag agaacccgat ccgtgctaagcccaggaacc 180 aacgtaaagc gctagcgtcc atgatgttcc tactaatcac cttcacgaataatccaaaca 240 ggcccacatc ttcccaagat caggatacag gttggtccaa aagtaaaagtcagggccggc 300 cctgtggggg agaaggcaag tgcccaggtt ctgatgggtg gcccagccctcagtctttca 360 cagaacgggt gcagatcaca ttagcactga tgggaaatag agatctcatgggacggtgaa 420 gacagagggc atacataagt agcaattgtg aaggtcggtt atcaatacgccccagaaaat 480 caaccatgat aatattccaa tctatgaaag catctgatac acttccacaaaggaagaagg 540 aaggaaatca tgggacctgc caaggtggac ttggcagagt gctaagagatgacccgattg 600 3 230 DNA Equus caballus 3 gtcgtagcgg agaaaggaatctctggattc catgcaatcc cagtcaaagt ggcagccata 60 tttgccggag agatagaagagagaatccta aagtgtctag ccagcaacaa gagcccctga 120 ataggccaag gaatcctcaggaaaacgaac aaagcaggac ggatcacact ttctgatttc 180 aacatagagg acgaagcgctaggtaccgaa acggaacagt ccgctacgac 230 4 285 DNA Equus caballus 4aaccgcggca tcgactagtc tcttcattgt cctaatgaga tccttctgga ctttggatta 60tgcttaaggc agaaggacac tgtaggtctg ataaggccca agtccggcct cgtgtttgca 120aacaagtttc aagattgaat cagcatggcc tccccataga cttgaacaga tgacaatgtt 180ctcctctcag tgcttgtccc tctcttctcg cttattgcca aggattcctt tcctatgcca 240agaatcagag tctgtttgat ctcttgcagt ttctgtgccg cggtt 285 5 230 DNA Equuscaballus 5 gtcgtagcgg agaaaggaat ctctggattc catgcaatcc cagtcaaagtggcagccata 60 tttgccggag agatagaaga gagaatccta aagtgtctag ccagcaacaagagcccctga 120 ataggccaag gaatcctcag gaaaacgaac aaagcaggac ggatcacactttctgatttc 180 aacatagagg acgaagcgct aggtaccgaa acggaacagt ccgctacgac230 6 217 DNA Equus caballus 6 kcggagggag gaatgtatgt attgcatgcaatcccagtca aagtggcagc catatttgcc 60 agagagatag aagagagaat cctaaagtttctagccagca acaagagccc ctgaataggc 120 caaggaatcc tcaggaaaac gaacaaagcaggacgtatca cactttctga tttcaaccta 180 aaggaccack cggtaggtac cgagacggatcgtccgc 217 7 220 DNA Equus caballus 7 gtagcggaga aaggaatctc tggattccatgcaatcccag tcaaagtggc agccatattt 60 gccagagaga tagaagagag aatcctaaagtttctagcca gcaacaagag cccctgaata 120 ggccaaggaa tcctcaggaa aacgaacaaagcaggacgta tcacactttc tgatttcaac 180 atagaggacg aagcgctagg taccgaaacggaacagtcgc 220 8 4693 DNA Equus caballus 8 gaattcatca aatttgccaaatttgtctcg gaaaagcatt gtgtccatga actggattgc 60 caggaccacc tcgaatttggagtttctgat ctcaggatgt caaagcaacc cagatgacca 120 cgggtactca gtgcttcgtttgacctaggc taggatagca aagagcacaa cgagtgacag 180 gtgcaaattc aaaaacagcaaccaagattc acgaaagcac tcacacctcg gcctagagaa 240 catacggtga ttcaagaaacacaaaattac tccctgggtg tgaaggagaa tggactgata 300 agcacatcaa agggagatagctgagaagac catgacgagt atactgtgga aaagaaaaaa 360 agggtccagg aaattgcaaggcgaacagat atcacacctg aataatccac gatgaaagat 420 gtcagttttt agacaggctctgctagaacg attgaaagtc agagaaagcc acaagccaaa 480 aaacggcagc ggaggtcaaacctgggctca ctgggaagtg tgagctatga gacagctcga 540 agcgggctct gaagccctttgtccatgacg attgccagga cagcagacac acacttatga 600 ccaatcctct gacgtcacagtggccccgac aaacaacctg gcatcctcta cacagtgcca 660 gagttgggct caagcagcatattcaatacc cattgcgctc aagtgcaggt tgccaatgga 720 acatgaaagg ggccggccggtggtgcagcg gtgaggtccg cagcttctga gtcagtggcc 780 aggggtgggc cgcgtggaatcctctgggtg agcctcctca ccactgcttc agtcattctg 840 ggccaggagt ccaagggggtgtagaggcag atgggaaggg acgttagata gggccagact 900 ttaacagcaa atacaagagcattgccagca gatgtgagca tagcgatgca cttctgccaa 960 aacacaacca attacaagttgcaaagaata gaaaaaggga agctaaaaac aaacaaagaa 1020 acgaaaaaca cagcttgccagtaccatgaa gaagatctct atgaagaacc ttagcaaaac 1080 aggtgaaagc tatgtcaatcgaaaaccagc aaacctttgg ggagagaaat tcaagaagac 1140 aaaaggaaaa agaaggatattttcagacct acgtatctaa gaattaacac actggagacg 1200 tccatagcgg agaaaggaatttctggattc catgcaatcc cagtcaaagt ggcagccata 1260 tttgccagag agatagaagagasaatccta aagtttctar ccagyracaa gagcccctga 1320 ataggccaag gaatcctcaggaaaaygaac aaagcaggac gtatcacact ttctgakttc 1380 aacatagagg acgaagcgctaggtaccgaa acggmacagt cgcgggctcc aaaacaggca 1440 cacagaccca tgcaacagaatcgagagagc agagactaac tcaaatatac atggacagcc 1500 catttgcgac cagggagccaagaggacaca gtggaccaag gattgtctct ccaataaact 1560 gtgctgggaa gcctgcatagccacaggaac acaacgagag tagaccatga tgttccacct 1620 ggcaaaggca ccacctgaaaaagattaaag ccctgaatgg cacacttgaa accgtgaaac 1680 ttttaggaga agacctaggcagagtgctct ttgccatctg tctgagccgc ctatttggaa 1740 gaagcctgtc tgacagggcaagggcagcaa aggagacaag aaacaaacgg gaccacctca 1800 aatgcagacg cttctgcccagtcaaggaaa ccatcgactc actgcaaaga cagcacaaca 1860 cctgggagtg gatgtttgcaaagcacacat cggacgaggg gtgaaaagcc cacagatgca 1920 atcaactcac acgtctccacaagaaaaaaa acaagcaaat gaaaacctgg gcaaaagatg 1980 gatacagaga tttctcccaataagatctaa agagggccaa caggttcatg aaaacttgtt 2040 cacccgcatt aagtcttagtccaatgcaaa tccaaaccgc aatgacatag cagctcactg 2100 tggtcagaat ggctataaggaggcccacag gaaaacaaca agtgtcagag aggaggtgga 2160 gagaagggaa gcctcctgcactgctggtgg cagtgtaaac gggtgcagcc actaggccaa 2220 gcggtgtgga actgcctcagaaatttaaga acccatgtcc cataggatcc agctattcct 2280 cggggccgtg tttagacaaagaactcggaa acacaaccgc taaagacatg tgcaccgctg 2340 agttcaccac agccttattcccgctctcca agacttggaa gccgcctggt gccgagcaag 2400 ggacgaatag agaaggacatgggctatagc cacacatggc ataccactca gcgggaacaa 2460 aggatgcaat ccagccatttgtgaccacca gaatggctgg gagggtttca gggtaagtga 2520 aaaaggcccc agggacatagtcaaataccg tagcatcaca cttacaagga gaagataaaa 2580 aaagcactaa ccaacaggtggcgcaggaca tttgattggg ggtgcccaga ggcaaagcgg 2640 ggcggtggcg agggtgtaagagatgatgag gcacaagtgt gtggtgaggt catgtgattc 2700 ggcttaagct ggtgaagaggatgtcaacta cacggaagtc caaatcgatg aggatgcaaa 2760 tctgaaagac ataggatattgtaacgcagg gttaaggcaa taaattcacc tacaatcaaa 2820 aatcccaaac gtgggtccagaatgacgaca tacagctctg ggagctaaaa gagattagag 2880 aaattgaaca tccaaggatggaaaatcacc tcaaaaggct tactgatgct acagaagggt 2940 agacccattc caaatccatgaagaagaatg cttaccaaga gaccttgatc cgtggttaag 3000 cccttggacc aaacccaaaacattaccgtc catgatgtta ctacttaata accttcacaa 3060 atcatccaaa caggcccacatcttcccaag ttcaggccac aggttggtcc aaaagtacaa 3120 gtcagggccg gccctgtggtggagtgggca agtgccctcg ttccgcttgg gtggcccggg 3180 cctcactctt tcacagcccgggtgcagatc acattagcac tgttaggaaa tagagatttc 3240 atgggacggt gcggacagaggtcataaaga agtagcaatt gtgagggtcc attatcagca 3300 cgccacagaa aaacaaccatgggaatatta caatctatga aggcatctgg tacacttccc 3360 caaaggaaga aggaaggaaaacatgggacc tgccaaggtg gacttggcag agtgctagga 3420 gatgacacga tcattgcgcatcagaggatt gcctgggcaa cttcaacttg ggagggagtc 3480 caggactttc tctggggaaggtccagtcac ttggccctct ccccaagaca taagatataa 3540 gagccagggt aatcttacagggaagaaacc agtgtctaga gtgaacggag caggcctttc 3600 caagttcgag gagaaagttagaaaccgaca agaaaatcag aaaaaggtaa actatcctac 3660 ctggaaaggg ggaggagtcagactggtctg agatggcact gaaactctgt gtgcttggag 3720 gttgtcagga caacttagccttctgaggtt ctttatgaca tcagggcgaa catgtccccc 3780 aagatggccg catggttacagtctgagtat tggcctaggc tgggctacta aaggatgaca 3840 tacccagaaa cagctttgacaaagagaaag cgggtacaga aactggaaga gatcaaacag 3900 actctgattc caggcataagaaagaaatcc ttggcaagaa gcgagaagag agggacaagc 3960 actgagagga gaacatcgtcatctattcaa gtctagggga ggccatgctg atgcaagcct 4020 gaaacttgat tggaaacacgaggtcggact tgggccttat cagacctaca gtgtccttct 4080 gccttaagca taatccaaagtcacgaagga tctcttgagg acattgaata ggagagtcga 4140 tgcctccttt cctaggcccctagcattctt tgaagatcag tctcactttc cataactctg 4200 gcgtcacggg ggcccactggatacatgcta atgcgtccca agaaatgtct tggaagcctt 4260 aaatgaatgg agccctgtcatgcttggggt aggtctcttt gttgggaacg gcctctccaa 4320 gtgtgctgaa aatcacccttttccagaggg cttggttcct ttgtgaaggc tgccctctca 4380 ggcttgtgtg ctcactttggctccaatgaa attctctccc cctacctctt cccgtatatg 4440 gattactgat tacgtgctttgacgccatat ggaattaagc tggctgaaaa ttagaacatt 4500 acaattctgt ttccagaaatatagacatgc cagggctgag gctgtaggtc aaacaaatgg 4560 cacacactat agacataaagtaagcccgta actagacgga atctagggca acgttcaact 4620 gtcaggggca agttcgaacctttccaaatc cacaaaaaag acagaaaaat atcattcctg 4680 gagagtggaa ttc 4693 93430 DNA Equus caballus unsure (1110) n at position 1110 = a, t, c, or g9 aagcttgccg ggacagcaga cacacaatta tgaccaatcc tctgacgtca cagtggcccc 60gacaaacaac ctggcctcct ctacactgtg ccagagttgg gctcaagcag catattccag 120ccacactgcg ctcaactgca ggtttccaat agaacatgaa agaggccgcc ggtggtgcag 180cggtgaggtc cgcagctttg tgtcggtggc ccgtggtggg ccaggtggaa tcctggggtt 240gagcctcctc accactgctt cagtcattct ggggcaggcg tccgagggcg tgtagaggct 300gatgggaagg gacgttagat agggccagaa ttccacagca aatacaggag cattgccggc 360agacgtgagc acatcgatgc acttctgcca aaacagaacc aataacaagt tgcaaagaac 420agaaaaacgg aagctaaaca aataaacaaa aataaacaca gcttcccagt acgaagaaga 480agatgtctat gaagaacctt agcaaaacag gtgaaagcta tgttaatcga aaatagcaaa 540tgtttgggga gagaaattta agaagacaca aggaaaaaga aggatattcc gcgacctagg 600aatggaagaa ttaacacact ggaaacgtcc atagcagaga aaggaatctc tggattccag 660gcaatcccag ttaaagtgga agccttcttt gccagarata tagaagagag aatcctaaag 720tttctagcca gcaagaagag cccctgaata ggccaaggaa tcctcaggaa aacgaacaaa 780gcaggacgta tcacactttc tgatttcaac atagagcacg aagcgctagg tactgaaacg 840gtacagtcca gtcccaaaaa caggcacaca gacaaatgta acactataga gagcccagag 900cccaactcaa acatacatgg acagcccatt tgcgaccagg gagccaagag gacacagtgg 960acaaacgaga gtctctccaa aaaacggtgc tgggaagcct gcacagccac atggtacaca 1020acgagagtag accatgatgt tccacctggc acacgcacca tctgaaattg attcaagccc 1080tgaatggcgc acttgaaacc cgggaacttn ttaggagaag acctaggcag agcgctctkk 1140cccatctgtc tgagccgccc acttggaaga agcctgtctg actcggcaag ggcagcaaag 1200gagacaagaa gcaaacggga ccacctcaaa tgcagacgct tctgcccagt caaggaaacc 1260atcgactcaa ggcaaagaca gcacaacaac tgggagtgga tgtttgcaaa gcacacatcg 1320gacgaggggt gaaaagccca aagatacaat caaatcacac gtctccacaa gaaataaaac 1380aagccaatga aaacctgcgc aaaagatgga cacagagact tctcccaaga agatctaaag 1440agggccaaca ggtgcatgaa aacttgttca ccctcattaa gtctgaggcc aatgcaaatc 1500caaaccgcaa tgacatagca gctaactgag ttcagaatgg ctataaggag accgacagga 1560cagcaacaat tgtcagaggg gaggtggaga gaagggaagc ctcctgcact gctggtggca 1620gtgtaaatgg gtgcagccac taaggcaagc aatgtngnac tggccacaga aatttaagaa 1680tccatgtcct ttaggatcca gcgattcctc gggggcgtgt ttagccaaag aaatcggaaa 1740ctcaacccgc taaagacatg tgcatcgctg agttcaccac agcttactcc gctctccaag 1800acttggaagc cgcctggtgc ccaacaagga agaatggaga agaacatggc tatagccaca 1860caatggcata ccactcattg cggaccagat gccatccagc cattgtggcc accagatggt 1920tgaggtttag gtgaagtgaa caggcccagg aaatagtgaa ttccatagca cgtcacttac 1980aaggagaaga aaaancaagc actaacccaa caggtggccc ttggacaatt tgattggggg 2040tgcccaaagg caaaaaaggg ccgttttgga aggagaaagg gatgatnagg cacaagtgcg 2100tggtgagtgc ctgtgattcg gcttacgctg gagaagagga tgtcgcctac acggaagtct 2160aaatcgatgg ggatgtaaat ctgaaagaca tcggatattc taatgcaggg ttatggtaat 2220aaattcacct acaatgaaaa atccaaaaag gggtccggaa tgacggcata cagctctggg 2280ggctgaaaga gattagagaa ggcgaacttt caaagatgga aaatcacctc aaaaggctta 2340ctgatgctac agaagggtag agcattccaa atacatgaag gggaatacta ccatgagaac 2400cttatccatg cttagcccag gaacccacgg aaaacgttac cgtccatgat gttactacta 2460atcaccttca cgaataatcc aaacaggccc acatcttccc aagttcaggc cacgggtcgt 2520ccaaaagtac aagtcaggga cggcgctgcg gggagcgggc aaatgcccat gttccgcttg 2580gtggcccggg cctcactctt tcacagcacg ggtgcagatc acattagcac tgttaggaaa 2640tagagatttc atggacrctc ggacaaaggk yataaatagg tagcaattgt gaagggtcag 2700ttatcaatac gccccagaaa agccaaccat gagaatattc caatctatga aggcatctgg 2760gtcactttcc acaaaggaag naggaagaaa acatgggacc tgccaaggtg gacttggaag 2820agtgctagga gatgacacgg taattgggca tcagaggact gcctgggcaa ttcaacttgg 2880aagggagtcc aggactttct gtggggaagg tccagccact tggccctttc cccaagacta 2940aagagataag agccagggtt atcttacagg gaagaaacca gtgtctagag agaatggagc 3000aggcctttcc aagttcgagg agaattttag aaacttacaa gaaataaaga aaaaggaaaa 3060caatcctacc tggaaagggg gaggagtcag actggtctga gatggctctg gaactctgtg 3120tgcttggagg ttgtcaggac aacttagcct tctgaggttg tttatgacat cacggtgaac 3180atgtccccca agatggccgc atggttatag tctgaggatt ggcctaggct gggctattaa 3240agggagacat acccaaaaac agctttgaca aacagaaagc gggcacagaa actggaagag 3300atcaaacaga ctctgattcc aggcataaga aaggaatcct tggcaagaag cgagaagaga 3360gggacaagca ctgagaggag aacattgtca tctgttcaag tctatgggga ggccatgctg 3420attcaagctt 3430 10 3450 DNA Equus caballus unsure (2122)..(2341) n atpositions 2122 through 2341 = a, t, c, or g 10 aagcttgccg ggacagcagacacacaatta tgaccaatcc tctgatgtca cagtggcccc 60 gacaaacaac ctggcctcatctacacagtg cgagagttgg gctcaagcag catattcaag 120 tcccattgcg ttcaactgcaggttgccaat ggaacatgaa acggccggcc ggtggtgcag 180 cggtgaggtc cgcagattctgggtcggtgg acaggggtgg gccgggtgga atcctggggg 240 tgaggctcct caccactgcttcagtcattc cggggcaggc gtccgcgtgg ttgtagaggc 300 agatgggaag ggacgttacatagggccaga cgtccacatc aaatacagga gcattgccag 360 cagatgtgag aacagcgatgcacttctgcc aaaacacaac caattacaag ttgaaaagat 420 tagaaaaacg gatgctaaaaacaaacaaac aaacaaaaaa cacagcttgc cagtacgaag 480 aagaagatct ctatgaagacacttagcaaa acaggtgaaa gctatgtcaa tagaaaacca 540 gcaaacgttt ggggaaagaaattcaagaag acacaaggaa aaagaaggat ataccgggac 600 ctaggaatgg aagaattaacacactggaaa cgaccatagc ggagaaggga atatctggat 660 tccattcaat cccagtcaaagtggcagcca tcttcgtcag agagatagaa gagagaatcc 720 taaagtttct agccagcaataagagcccct gaataggcca aggaatcctc aggaaagcaa 780 acaaagcaga acgtatcacactttctgatt tcaacataga ggacgaagcg ctaggtaccg 840 acacggcaca gtccgggcccaaacacagac acacagaccc atgcaacaga atcgagagcc 900 cagagcccaa ctcaaacatacatggacggc ccatttgcga ccaaggagcc aagaggagac 960 agcggacaaa ggagagtctctccaataaac gctgctggga agtctgaaca gccacatggt 1020 gcacaacgag agtagaccatgatgttccac ctggcacacg cagaacctga aatggattaa 1080 agccctgaat ggcacacttgaaaccgtgaa acttgtagga gaagacctag gcagagtgct 1140 ctttgccatc tgtctgagccacctatttgg aagaagcctg tctgactggg caagggcagc 1200 aaaggagaca agaaacaaacgggaccacct caaatgcaga cgcttctgcc cagtcaagga 1260 aaccatcgac tcaatgcaaagacagcacaa caactgggag tggatgtttg caaagcacac 1320 atcggactaa gggtgaaaagcccaaagata caatcaactc acacgtctcc acaagaaaaa 1380 aaacaagcca atgaaaacctgggcaaaaga tggacacaga gatttctccc aagaagatct 1440 aaagagggcc aacaggtgcatgaaaacttg ttcaccctca ttaagtctga ggccaatgca 1500 aatccaaacc gcaatgacatagcagctcac tgtggtcaga atggctataa ggagtccgac 1560 aggaaaacaa ttgtcagagaggaggtggag agaagggaag ccgcctgcac tgctggtggc 1620 actgtaaacc ggtgcagccactatgccaag cggtgtggaa ctgcctcagg aatttaagaa 1680 tccatgtccc ataggatgccgctattcctc gggggcgtgt ttagccaaag aactcggaaa 1740 cacaaccgcc taaagacatgtgcaccgctg agttccccac agccttactc ccgctctcca 1800 agacttggaa gccaccctggtccccagcaa gggacgaatg gagaaggaac atgggctata 1860 gccacacaat ggcaaaccactcagcgggaa caaaggatgc aatccagcca tttgtgagca 1920 ccagaatggc tgggaggcttttaggggaag tgaaacaggc cccagggaca tagtcaaata 1980 ccgtaggatc tcactttcaaggagaagata aaagaagaac taaccaacag gtggcgctgg 2040 acatttgatt gggggtgcccagaggcaaag cggggccatg ggggaggagt gacagagata 2100 gatgaggcaa atgggtgtgacnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 2340 nyacntcaaa aggcttactgatgcgacaga agggtagcca ttccaaatac atgtagagga 2400 atactaccaa gagaacctgatccatgctaa acccaggaac caacggaaaa tgttaccgtc 2460 catgatgttg ctactaatcaccttcaggaa taatccaaac aggcccacaa cttccccaag 2520 ttcaggccac aggttggtccaaagtcaagt ccagtttcgg ccctgtggtg gatcgggcaa 2580 gtgaccacgt tccgcttgggtggcccgggc ctcactcttt cacagcacgg gtgcagatca 2640 cattagcact gtaaggaaatagagatttca tgggacggtg cggacagagg tcataaagaa 2700 gtagcaattg tgagggtccgttatcaatac accccagaaa agcatccatt agaatattcc 2760 aatctatgaa ggcatctggtacacttcccc aaaggaagaa ggaaggaaaa catgggacct 2820 gccaaggtgg acttggcagagtgctaggag atgacaggat cattgggcat ctgaggatta 2880 cctgggcaac ttcaacttgggatggagtcc aggactttct ctggggaagg tccagccact 2940 tggccctctc cccaagacataagagataag agccagggta atcttacagg gaagaaacca 3000 gtgtctagag agaatggagcaggccattcc aagttcgagg agaaagttag aaaccgacaa 3060 gaaatccaga aaaaggaaaactatcctacc tggaaagggg gatgagtcag actggtctga 3120 gatggctctg aaactctgtgtgcttggagg ttgtcaggac aacttagccg tctgaggttg 3180 tttgtgacat cacggtgaacatgtccccca agatggaggc atggttacag tctgagcatt 3240 ggcctagcct gggctattcaagggggacat acccaaaaac agctttgaca aacagaaagc 3300 gggcacagaa actggatgagatcaaacaga ctctgattcc aggcataaga agggaatcct 3360 tggcaagaag cgagaagagagggtcaagca ctgacaggag aacatttccc tctgttcaag 3420 actataggga ggccatgctgattcaagctt 3450 11 4344 DNA Equus caballus 11 aagcttgaat cagcatggcctccccataga cttgaacaga tgacagtgtt ctcctctcag 60 tgcttgtccc tctcttctcgcttcttgcca aggattcctt tcttatgcct ggaatcagag 120 tctgcctgat ctcctccagtttctgtgccc gctttctgtt tgtcaaagct gtttctgggt 180 atgtctccct ttaatagcccagcctaggcc aatcctcaga ctgtaaccat gcggccatct 240 tgggggacat gttcaccgtgatgtcataaa caacctcaga aggctaagtt gtcctgacaa 300 cctccaagca cacagagtttcagagccatc tcagaccagt ctgactcctc ctcctttcca 360 ggtgattact agtaacatcatggagggtag cctttcagtt tgttcctgga cttagcactg 420 atcaggttct ctttgtagtattcctcttca tgtatctgga atggtgtacc cttctgtcgc 480 atcagtaaga cttttcaggtgattttctgg tacacttccc caaagcaaga aggaaggaga 540 acttgggacc tgccaaggtggtcttggcag agtgctagga gatgagaaca aaggatgcaa 600 tccagccatt tgtgaccatcagaatgggtg ggagggtttt aggggaagtg aaacaggccc 660 cagggacata gtcaaacaccataggattca ctttcaagga gaagatataa gaagcactaa 720 ccaacaggtg gcgctggacatttgattggg ggtgcccaga ggcaaagccg ggcagtgggg 780 gagggtgaaa gagatgatgaggcacaagtg tgtggtgagg gcctatgatt cggcttacgc 840 tggggaagag gatgtcacctacacggaagt ctaaatcgat gaggatgtaa atctgagaga 900 cataggatgt tctaatgcagggttatggca ataaattcac ctacaatcaa aaatccaaaa 960 agtggtccag aatgactccataaagctctg ggggctgaaa gagattagag acggtgaact 1020 tcaaaggatg gaaaatcacctcaaaagtct tagtgatgcg acagaagggt agaccattcc 1080 aaatacatga agaggaatactacaaagaga acctgatccg tgctaagccc aggaaccaac 1140 ggaaaatgtt accgtccatgatgttactac taatcacctt catgaataat ccaaacaggc 1200 cctcatcttc ccaagatcaggccacaggtt ggtccaaaag tacaagtcag ttccggctct 1260 gtggtggagc gggcaagtgcccacgttccg cttgggtggg cagggcttca gtctttcaca 1320 gcacgggtca gatcccattagccttgttag gaaatagaga tttcttggac ggtcggacag 1380 aggtcataaa gggacgttagataaggcaga tttccacagc aaatacagga gcattcccag 1440 cagatgtgat cacaacgatgcacttctgcc aaaacacaac cacttacaag ttgcaaagat 1500 tagaaaaccg gaagctaaaaacaaaaaaac aaaaacaaaa cacagcttgc cactacgaag 1560 aagaagatct ctatgaagaaccttagcaaa acaggtgaaa ggtatgtcaa tctaaaacca 1620 acaaacgttt ggggaaagaaattcaagaag acacaaggaa taagaaggat attccgggac 1680 ctaggaatgg aagaattagcacacttgaaa cgtccataga ggagaaagga atctctggat 1740 tccatgcaat cccagtcaaagtggcagcca tatttgccag agagatagaa gagagaatcc 1800 taaagtttct agccagcaagaaaagcccct gaataggcca aggaatcctc aagaaaacga 1860 acaaagcagg acggatcacactttctgatt tcaacataga ggacgaagcg ctaggtactg 1920 aaacggcaca gtccgggcccaaaaacaggc acacagaccc atgcgacaga atcgagagcc 1980 cacagcccaa ctcaaacatacatggacacc ccatttgcga ccagggagcc aagaggagac 2040 agtggacaaa ggagagtctctccaataaac gggctgggaa gcctgacagc cacatagaac 2100 acaagaatag accatgatgttccacctggc agacgcccac tgaatggatt caagccctga 2160 tggccacttg aaccgtgaacttgtaggaga agacctagca gagtgctctt tgccatctgt 2220 ctgacccgcc tatttggaagcaggctgtct gactgggcaa gggcagcaaa ggagacaaga 2280 aacaaacggg accacctgaaatgcagacgc ttctgcccag tcaaggaaac catcgactca 2340 atgcaaagac agcacaacacctgggagtgg atgttagcaa agcacacatc ggatgagggg 2400 cgaaaagccc aaagatacaatcaactcaca cgtctccaca agaaaaaaaa caagccaatg 2460 aaaatctggg caaaagatggacacagagat ttctcccaag aagatctaag agggccaaca 2520 ggtgcatgaa aacttgttcaccctcattag tctaaggcca atgcaatcca accgcaatga 2580 catagcagct cacttgtggtcagaatggct ataaggaggc agacaggaaa acaacaagtg 2640 tcagagagga ggtggagagtaggaagcctc ctgcactgct ggtggcagtg taaacgggtg 2700 cagccagtag gccaagcggtgtgaaactac ctcagcaatt tcagaatccg tgtaccatag 2760 gatccagcta ttcctcgggggcgtgtttag ccaaaaaact cggaaacaca aactcctaaa 2820 gacatgtgca ccgctgagttcaccacagcc ttactcccgc tctccaagac ttggaagccg 2880 tcctggtgcc gagcaagggacgaatgtaga aggaacatgg gctatagcca cacaatggca 2940 taccagtcag tgggaacaaaggttgcaatc cagtcatttg cgaccaccag aatggcttgg 3000 agagttttat gggaagtgaaacaggaccca gggatatagt caaataccgt agcataacac 3060 ttacaaggag aagataaaaaaagcactaac caacaggtgg cgctggacat ttgattgggg 3120 gtgcccagag gcaaagcggggcggtggggg agggttaaag acttgatgag gcacaagtgt 3180 gtggtgagga catgtgattcggcttatgct ggtgaagagg atgtcaccta cacggaagac 3240 taaatcgatg agcatgtaaatgtgaaagac atagggtgtt ctaatgcaag gttatggcac 3300 taaattcacc tacaatcaaaaatccaaaaa ggggtccaga atgacggcat acagctctgc 3360 gggctgaaag agattagagaaggtgaactt ccaaggatgg aaaatcacct caaaaggctt 3420 actgatgcta cagaagggtgaccattccaa atacttgaag aggaatacta ccaagagaac 3480 ctgatcagtg ctaagcccaggaaccaacgg aaaacgttac catccatgat gttactacta 3540 atcaccttcg cgaataatccaaacaggccc acatcttccc aagttcaggc cacatgttgg 3600 tccaaaagta caagtcagggaagccctgtg ggggagcggg caagggccaa cgttccgctt 3660 gggtggccag ggcctcactctttcacagca cgggtgcaga tcacattagc actgttagga 3720 aatagagatt tcatgggacggtgcggacag aggacataaa gaagtagcaa ttgtgagggt 3780 cctttatcaa tacgccccaaaaaagcaacc atgacagtat tccaatctat gaaggcatct 3840 ggaacacttc cccaaaggaagaaggaagga aatcatggga cctgccaaga tggacttggc 3900 agagtgctag gagatgactggatcattgca catcagagga ttgcctgggc aacatcaact 3960 tgggagggag tccaggactttctctgggga aggtccagcc acttatccct ctctccaaga 4020 cataagagca agagccagggtatcttacag ggaagaacca gtgtctagag agaatggaca 4080 accctttcca agttcgaggataaagttcga aaccgacaag aaatccagaa aaaggaaaac 4140 tatcctacct ggaaagggggaggagtcaga ctggtctgag atggctctga aactctgtgt 4200 gctgggaggt tgtcaggacaacttagactt ctgaggttgt ttatgacata acggcgaaca 4260 tgtcccccaa gatggccgcatggttactgt gtgaggattg gcctaggctg ggctattaaa 4320 ctgagacata cccagaaaaagctt 4344 12 21 DNA Equus caballus 12 agcggagaaa ggaatctctg g 21 13 22DNA Equus caballus 13 tacctagcgc ttcgtcctct at 22 14 23 DNA Equuscaballus 14 ttcgtcctct atgttgaaat cag 23 15 21 DNA Equus caballus 15gtcgtagcgg agaaaggaat c 21 16 20 DNA Equus caballus 16 agcggactgttccgtttcgg 20 17 21 DNA Equus caballus 17 gcccagtgtt tcgttggttc g 21 1825 DNA Equus caballus 18 catagttgta tattcttcgt tgtgg 25 19 41 DNA Equuscaballus 19 gcggtcccaa aagggtcagt agcggagaaa ggaatctctg g 41 20 42 DNAEquus caballus 20 gcggtcccaa aagggtcagt tacctagcgc ttcgtcctct at 42 2141 DNA Equus caballus 21 gcggtcccaa aagggtcagt gcccagtgtt tcgttggttc g41 22 45 DNA Equus caballus 22 gcggtcccaa aagggtcagt catagttgtatattcttcgt tgtgg 45 23 10 DNA Equus caballus 23 ccagaacgga 10 24 10 DNAEquus caballus 24 caatcgggtc 10 25 10 DNA Equus caballus 25 gtcgtagcgg10 26 10 DNA Equus caballus 26 tcacgtacgg 10 27 10 DNA Equus caballus 27aaccgcggca 10 28 23 DNA Equus caballus 28 atttaggtga cactatagaa tac 2329 23 DNA Equus caballus 29 attatgctga gtgatatccc gct 23

What is claimed is:
 1. An isolated polynucleotide, the polynucleotidehaving a sequence as set out in any one of SEQ ID NOS: 1 to 4 or 8 to11, or a sequence which hybridises thereto under high stringency,wherein the polynucleotide hybridises specifically to the equine Ychromosome.
 2. An isolated polynucleotide, the polynucleotide having asequence containing nucleotides 990-2497 of SEQ ID NO: 8, 421-1920 ofSEQ ID NO. 9, 421-1930 of SEQ ID NO.
 10. or 1502-2996 of SEQ ID NO. 11or a sequence which hybridises thereto under high stringency, whereinthe polynucleotide hybridises specifically to the equine Y chromosome.3. An isolated polynucleotide as claimed in claim 1 in which thesequence shares at least 60% homology with a sequence shown in any oneof SEQ ID NOS 1 to 4 or 8 to
 11. 4. An isolated polynucleotide asclaimed in claim 3 in which the sequence shares at least 80% homologywith a sequence shown in any one of SEQ ID NOs 1 to 4 or 8 to
 11. 5. Anisolated polynucleotide as claimed in claim 1 which has a sequence asset out in SEQ ID NO: 3 or a sequence which hybridises thereto underhigh stringency.
 6. A vector including a polynucleotide sequence asclaimed in claim
 1. 7. A host cell including a vector as claimed inclaim
 6. 8. An oligonucleotide probe or primer of at least 18nucleotides that hybridizes specifically to the equine Y chromosome, theoligonucleotide having a sequence that hybridises to a polynucleotide asclaimed in claim
 1. 9. An oligonucleotide probe or primer as claimed inclaim 8 which includes a sequence selected from: AGCGGAGAAAGGAATCTCTGG,(SEQ ID NO: 12) or TACCTAGCGCTTCGTCCTCTAT (SEQ ID NO: 13).
 10. Anoligonucleotide probe as claimed in claim 8 in which the probe isconjugated to a detectable label.
 11. An oligonucleotide probe asclaimed in claim 10 in which the label is selected from a radioisotope,an enzyme, biotin, a fluorescer or a chemiluminescer.
 12. A method ofdetermining the sex of a horse, an equine fetus, an equine embryo or anequine cell(s) which method includes analysing a biological samplederived from the horse, fetus, embryo or cell(s) for the presence of apolynucleotide sequence as set out in any one of SEQ ID NOS: 1 to 4 or 8to 11, wherein the presence of the polynucleotide in multiple copynumber is indicative that the biological sample is derived from a male.13. A method according to claim 12 wherein the multiple copy number isgreater than 5 copies in the haploid genome.
 14. A method according toclaim 12 in which the biological sample includes one or more spermcells.
 15. A method according to claim 12 in which the biological sampleincludes nucleated fetal cells.
 16. A method according to claim 12 inwhich the analysis involves Southern blot hybridisation, dot blothybridisation or in situ hybridisation.
 17. A method according to claim16 in which the analysis involves the use, as a probe in the Southernblot hybridization, dot blot hybridization or in situ hybridization, ofan oligonucleotide probe that hybridizes specifically to the equine Ychromosome, the oligonucleotide having a sequence that hybridises to apolynucleotide having a sequence as set out in any one of SEQ ID NOS: 1to 4 or 8 to 11, or a sequence which hybridises thereto under highstringency, wherein the polynucleotide hybridises specifically to theequine Y chromosome.
 18. A method according to claim 12 in which theanalysis involves the polymerase chain reaction or ligationamplification reaction.
 19. A method according to claim 18 in which theanalysis involves the use, as a primer in the polymerase chain reactionor ligation amplification reaction, of an oligonucleotide primer thathybridizes specifically to the equine Y chromosome, the oligonucleotidehaving a sequence that hybridises to a polynucleotide having a sequenceas set out in any one of SEQ ID NOS: 1 to 4 or 8 to 11, or a sequencewhich hybridises thereto under high stringency, wherein thepolynucleotide hybridises specifically to the equine Y chromosome.
 20. Akit for sex determination of a horse, an equine fetus, an equine embryo,an equine cell or a population of equine cells, which includes apolynucleotide as claimed in claim 1 or an oligonucleotide probe orprimer of at least 18 nucleotides that hybridizes specifically to theequine Y chromosome, the oligonucleotide having a sequence thathybridises to a polynucleotide having a sequence as set out in any oneof SEQ ID NOS: 1 to 4 or 8 to 11, or a sequence which hybridises theretounder high stringency, wherein the polynucleotide hybridisesspecifically to the equine Y chromosome.
 21. An isolated polynucleotideas claimed in claim 1, wherein the polynucleotide is less than 500nucleotides in length.
 22. An isolated polynucleotide as claimed inclaim 2, wherein the polynucleotide is less than 500 nucleotides inlength.